PT835315E - Vaccine for porcine reproductive and respiratory syndrome virus - Google Patents

Abstract

The present invention provides a purified preparation containing, for example, a polynucleic acid encoding at least one polypeptide selected from the group consisting of proteins encoded by one or more open reading frames (ORF's) of an Iowa strain of porcine reproductive and respiratory syndrome virus (PRRSV), antigenic regions of such proteins which are at least 5 amino acids in length and which effectively protect a porcine host against a subsequent challenge with a PRRSV isolate, and combinations thereof in which amino acids non-essential for antigenicity may be conservatively substituted. The present invention also concerns a polypeptide encoded by such a polynucleic acid; a vaccine comprising an effective amount of such a polynucleic acid or protein; antibodies which specifically bind to such a polynucleic acid or protein; methods of producing the same; and methods of protecting a pig against a PRRSV and treating a pig infected by a PRRSV.

Description

DESCRIPTION

The present invention relates to polynucleic acids isolated from a porcine reproductive and respiratory syndrome virus (RSVRS), a protein and / or a polypeptide encoded by polynucleic acids, a vaccine that protects the virus from the reproductive and respiratory syndrome of pigs. the pigs of a PRRSV using protein or polynucleic acids, processes for the production of proteins, polypeptides and polynucleic acids, an antibody to the polypeptides, the use of the antibody in the preparation of a medicament for the treatment of pigs with SRRS (reproductive syndrome and respiratory tract of pigs), a vaccine production process and a process for the detection of RSVRS. The Swine Reproductive and Respiratory Syndrome (SRRS), a new and serious disease in pigs, was first reported in the United States in 1987 and was rapidly recognized in many Western European countries (reviewed by Goyal in the Journal of Veterinary Diagnostic Investments , 1993, 5: 656-664, and in U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435). The disease is characterized by reproductive failures in sows and pigs, pneumonia in young growing pigs, and increased mortality in piglets in the preweaning period (Wensvoort et al., Vet., 13: 121-130, 1991 ; Christianson et al., 1992 Am. J.

Vet Res 53: 485-488; U.S. Patent Applications Serial Nos. 08 / 131,625 and 08 / 301,435). The SRRS-causing agent, the reproductive and respiratory syndrome virus (RSVRS) was first identified in Europe and later in the United States (Collins et al., 1992, J. Vet. Diagn. Invest., 4: 117-126). The European strain, designated as Lelistad virus (VL), was cloned and sequenced (Meulenberg et al., 1993, Virology, 192: 62-72 and J. Gen. Virol., 74: 1697-1701; Conzelmann et al., 1993 , Virology, 193: 329-339). RSVRS has been provisionally classified into a new proposed family of Arteriviridae viruses, including equine arthritis virus (VAE), lactic dehydrogenase-enhancing virus (VDHL) and simian hemorrhagic fever virus (VFHS) (Plagemann and Moennig, 1992, Adv. Virus Res., 41: 99-192; Godeny et al., 1993, Virology, 194: 585-596, U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435). This group of viruses, with simpler helical structure RNA, has many common features, such as genome organization, replication strategy, macrophage morphology and tropism (Meulenberg et al., 1993; with Serial Nos. 08 / 131,625 and 08 / 301,435). Subclinical infections and persistent viraemia, with simultaneous production of antibodies, are also one of the histopathological features of arteriviruses.

Genetic and pathogenic antigenic variations have been reported in the different isolates of the RSVRS (Wensvoort et al., 1992, J. Vet Diagn. Invest., 4: 134-138; Mardassi et al., 1994, J. Gen. Virol., 75: 681-685, U.S. Patent Applications Serial Nos. 08 / 131,625 and 08 / 301,435). In addition, the RSVRS of the United States and Europe have two different genotypes (applications for 2 U.S. patents Nos. 08 / 131,625 and 08 / 301,435). Antigenic variability also exists among the different isolates of the North American virus (Wensvoort et al., 1992). Significant differences in pathogenicity have already been observed not only between the isolates of the viruses from the United States and Europe, but also between the different isolates of the North American viruses (U.S. patent applications Serial Nos. 08 / 131,625 and 08 / 301,435). The organization of the arterivirus genome resembles that of the coronaviruses and toroviruses insofar as their replication involves the formation of a set of subgenomic mRNAs nested at the common 3 'end (sg mRNAs) (Chen et al., 1993, J. Gen. Virol, 74: 643-660, Den Boon et al., 1990, J. Virol., 65: 2910-2920, De Vries et al., 1990, Nucleic Acids Res., 18: 3241-3247, Kuo et al. 1991, J. Virol., 65: 5118-5123, Kuo et al., 1992, U.S. Application Serial Nos. 08 / 131,625 and 08 / 301,435). Partial sequences of several U.S. isolates have also been determined (U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435; Mardassi et al., 1994, J. Gen. Virol., 75: 681 -685). The genome of the PRRSV is polyadenylated, about 15 kb in length and contains eight open reading frames (SLQs) (Meulenberg et al., 1993; U.S. Patent Applications Serial Nos. 08 / 131,625 and 08 / 301,435). The QLAs 1a and 1b probably encode the viral RNA polymerase (Meulenberg et al., 1993). It was found that QLAs 5, 6 and 7 respectively encoded a glycosylated membrane (E) protein, a non-glycosylated membrane protein (M) and a nucleocapsid (N) protein 3 (Meulenberg et al., 1993). QLAs 2 to 4 appear to have the characteristics of membrane-associated proteins (Meulenberg et al., 1993; U.S. Application Serial No. 08 / 301,435). However, the translation products of QLAs 2 to 4 were not detected in the lysates of virus-infected cells or in the virions (Meulenberg et al., 1995).

The VAE envelope major glycoprotein encoded by QLA 5 may be the virus binding protein and the neutralizing monoclonal antibodies (AMNs) are targeted to this protein (de Vries, J. Virol, 1992; 66: 6294-6303; Faaberg , J. Virai, 1995, 69: 613-617). The primary envelope glycoprotein of VDHL, a virus very similar to the RSVRS, is also encoded by QLA 5 and it was found that several different neutralizing monoclonal antibodies immunoprecipitated the QLA 5 protein (Cafruny et al., Vir. Res., 1986; 5: 357-375).

Thus, the primary envelope protein of the RSVRS, encoded by QLA 5, is likely to induce neutralizing antibodies against RSVRS.

It has been suggested that the antigenic variation of the virus is the result of the direct selection of variants made by the host immune response (reviewed by Domingos et al., J. Gen. Virol 1993, 74: 2039-2045). Thus, it is likely that these hypervariable regions are due to selection pressure of the host immune response and may explain the antigenic diversity observed in the RSVRS isolates.

The M and N proteins of the United States RSRRS isolates, including ISU 3927, are highly conserved (U.S. Serial No. 08 / 301,435). The M and N proteins are integral to preserve the structure of the RSVRS virions and the N protein may be subject to stringent functional restrictions. Therefore, (a) the M and N proteins are not subject to a high antibody selection pressure or (b) the QLAs 6 and 7, which probably encode the M and N proteins, are responsible for the virulence of the viruses or are correlated with it. Interestingly, however, a greater variation of the VLDH M protein sequence was observed between VLDH isolates with different neurovirulence (Kuo et al., 1992, Vir Res. 23: 55-72).

LFSs 1a and 1b are predicted to translate into a single protein (viral polymerase) by changing the reading frame sequence. QLAs 2 to 6 can encode the viral membrane associated with proteins.

In addition to the genome RNA, many animal viruses produce one or more sg mRNA species to allow expression of the virus genes in a regulated fashion. In the RSVRS-infected cells, seven species of virus-specific mRNAs are synthesized representing a set 3 'common end nested (mRNAs 1 to 7, in descending order of size). MRNA 1 represents the mRNA of the genome. Each of the sg mRNAs contains a leader sequence derived from the 5 'end of the virus genome.

The number of sg mRNAs differs between arteriviruses and even between different isolates of the same virus. A nested set of 6 sg mRNAs was detected in the cells infected by VAE and cells infected by the European RSVRS. However, in VDHL infected cells, a set of 6 sg (VDHL-C) or seven mRNAs (VDHL 5-P) is present in addition to the RNA in the genome. The additional sDNA-1 mRNA of the VDHL-P contains the 3 'end of the QLA1 and may potentially be translated to a protein present at the C-terminus of the viral polymerase. Analysis of the sg mRNA sequence of VDHL and VAE indicates that the leader mRNA junction element is conserved. It has also recently been found that the junction sequences of the European VL leader mRNA contain a common motif, UCAACC or a very similar sequence.

Sg mRNAs packaged in coronavirus virions, such as bovine coronavirus (CoVB) and transmissible gastroenteritis virus (VGET), were found. However, only traces of sg mRNAs were detected in purified virions of the rat hepatitis virus (VHR), another coronavirus. VDHL sg mRNAs, an element very close to the RSVRS family, are also not packaged in the virions and only gnomic RNA was detected in the purified VDHL virions.

The sg mRNAs of VDHL and VAE have already been characterized in detail. However, information regarding sg mRNAs of the RSVRV strains, especially the U.S. RSVRS, is very limited. The need for molecular characterization of sg mRNAs from the U.S. RSVRS was thus realized. The VHR packaging signal is located at the 3 'end of the QLA 1, so only the genomic VHR RNA is packaged. The sg mRNAs of VCB and VGET were, however, found in the purified virions. The packaging signal of VCB and VGET has not been determined. The alphavirus Aura sg mRNA is indeed packaged in the virions because presumably the packaging signal is present in the mRNA sg. Sindib virus 6 sg 26S mRNA is not packaged in the virions because the packaging signal is located in the genome segment (not present in sg mRNA). The sg mRNAs of VDHL, a closely related element with the RSVRS, are also not packaged in the virions. There are many mechanisms involved in the formation of sg. It has been suggested that coronaviruses utilize a single transcription mechanism initiated by the leading RNA in which the leading RNA is transcribed from the 3 'end of the genome-sized negative helix array RNA, then dissociates from the template and then joins the RNA from the matrix downstream of the intergenic regions to initiate transcription of the mRNAs sg. The template predicts that the 5 'leader contains a specific sequence at its 3' end which is further downstream in the genome preceding each of the A2Bs. The leader joins the body of each sg mRNA through of the mRNA junction segment to the leader. RSVRS is one of the important causes of pneumonia in newborn pigs and weaning pigs. SVRR causes significant economic losses in pig farms (the extent of such losses is not fully known). Reproductive diseases have been the main consequence of RSVRS infections in recent years due to the early prevalence of RSVRS strains with relatively low virulence. Respiratory diseases have now become the main problem associated with RSVR, due to the increasing prevalence of RSVRS strains with relatively high virulence. There was a need to create a vaccine to protect against diseases caused by the various strains of RSV. 7

Interestingly, in the United States and the rest of the world the market for vaccines for animals is larger than the market for vaccines for humans. There is thus an economic incentive to develop new veterinary vaccines, as well as the great public health benefit of protecting farm animals from disease.

Accordingly, it is an object of the present invention to provide a polynucleic acid isolate from the reproductive and respiratory swine syndrome virus (RSVR), VR2430, which encodes a PRRSV protein.

It is also an object of the present invention to provide a PRRSV protein either isolated from a PRRSV or encoded by PRRSV polynucleic acid.

It is also a further object of the present invention to provide a vaccine based on a protein or a polynucleic acid which protects the pigs against RSVRS.

It is still a further object of the present invention to provide a method of detecting RSVP.

It is yet another object of the present invention to provide an antibody that immunologically binds to a PRRSV protein or to an antigenic region of that protein.

It is a further object of the present invention to provide the use of the antibody in the manufacture of a medicament for the treatment of a pig with SRRS.

It is still another object of the present invention to provide a screening or detection kit for RSVS.

This and other objects which will appear in the description of the preferred embodiments described below were provided by a purified preparation according to claim 1 below. The present invention also provides a purified polypeptide according to claim 8, vaccines as claimed in claims 9 and 10, an antibody as claimed in claim 13, a diagnostic kit as claimed in claim 14, a method for producing a polypeptide as claimed in claim 15 and an expression vector containing the polynucleic acid under the operational control of a promoter as claimed in claim 16.

The following is a description by way of example only with reference to the drawings accompanying the embodiments of the present invention. In the drawings: Figure 1 shows a comparison of the nucleotide sequence of QLAs 2 to 5 of the isolates VR 2474 (ISU 79), VR 2475 (ISU 1894), VR 2431 (ISU 3927), VR 2429 (ISU 22) and VR 2430 (ISU 55) with other known isolates of the VSRRSS;

Figures 2A, 2B and 2C respectively show the alignment of the deduced amino acid sequences of QLA 2, QLA 3, QLA 4 and QLA 5 of VR 2474 (ISU 79), VR 2475 (ISU 1894), VR 2429 (ISU 22 ), VR2 430 (ISU 55) and VR2431 (ISU 3927) from the US with other known isolates from the VSRRSS; Figure 3 shows a phylogenetic tree based on the nucleotide sequences of the BLAs 2 to 7 of seven isolates with different virulence of the RSVRS of the US Figure 4 shows a Northern blot analysis of RNA isolates from infected CRL 11171 cells by VR 2431 (1SU 3927) (column 1) and purified virions of VR 2431 (ISU 3927) (column 2); Figure 5 shows a " Northern blot " of isolates of total intercellular RNAs from CRL 11171 cells infected with VR2429 (ISU22) (column 1), VR 2430 (ISU 55) (column 2), VR 2474 (ISU 79) (column 3), VR 2475 (ISU 1894) ( column 4) and VR 2431 (ISU 3927) (column 5), respectively;

Figures 6A and 6B show Northern blotting of total RNA isolates from CRL 11171 cells infected with VR 2474 (ISU 79) with different values of the multiplicity of infection (mdi) (A) and polyadenylated RNA from cells infected with the isolates of VSRRS VR 2430 (ISU 55) and VR 2474 (ISU 79) (B);

Figures 7A and 7B show Northen blot analysis of isolates of total intercellular mRNAs from CRL 11171 cells infected with VR 2475 (ISU 1894) (A) and VR 2474 (ISU 79) (B);

Figures 8A and 8B show PCR-TR (polymerase chain reaction-reverse transcriptase) amplification of the 5 'end sequences of ISU 1894 sg and 4 mRNAs (column 1) and sg 3, 4 and 4 mRNAs -1 of VR 2474 (ISU 79) (column 2) (A) wherein column L is a 1 Kb marker; and the mRNA-junction sequences leading to the mRNAs sg 3 and 4 of VR 2474 (ISU 79) and VR 2475 (ISU 1894) and mRNA sg 4-1 of VR 2474 (ISU 79) (B), wherein the locations of the leading mRNA-junction sequences in the genomes relative to the start codon of each QLA were indicated with the (-) sign followed by the number of nucleotides upstream of the QLAs; and Figure 9 shows the alignment of the sequence of QLAs 2 to 7 of VR 2475 (ISU 1894) and VR 2474 (ISU 79), wherein the initial codon of each QLA is indicated by + > signals, the termination codon of each QLA is indicated by an asterisk (*), the leading or trailing mRNA junction sequences are underlined, and the locations of the leading mRNA junction sequences relative to the initiation codon of each QLA are indicated with the signal (-) followed by the number of nucleotides upstream of each of the QLAs.

In the present application, the nucleotide sequences of QLAs 2 to 5 of a low virulence isolate and four other isolates of RSVRS of the Iowa strain with 'moderate' and high virulence were determined. Based on comparisons of QLAs 2 to 7 of several RSVRS isolates, the less virulent US isolate, known as VR 2431 (ISU 3927), has relatively high sequence variations in QLAs 2 to 4 compared to other variations in other isolates isolates from the US In addition, based on the analysis of QLA sequences, at least three minor genotypes exist in the main genotype of the US RSVRR

Analyzes of the QLA 5 protein sequences from different RSRSV isolates reveal the existence of three hypervariable regions containing non-conserved amino acid substitutions. These regions are hydrophilic and also antigenic as predicted by computational analysis.

In the present invention, a "reproductive and respiratory syndrome virus of the swine" or "RSVRS" refers to a virus which causes SRRS, RAAS (respiratory syndrome and epidemic epidemic of pigs), SSRI (infertility and respiratory pigs), DMP (Mysterious Pigs Disease) and / or LIV (PIP in the English terminology, which has been abandoned), including the Iowa strain of RSVRS, other RSVRS strains found (eg VR 2332), RSVRV strains found in Canada (eg, IAF-exp91), RSVRR strains found in Europe (eg, Lelystad virus, RSVRS-10) and variants very similar to these viruses that have appeared or will appear in the future. The "strain of Iowa" of the RSVRS includes (a) the isolates of the RSVRS deposited with the American Type Culture Collection by the applicants of the present application and / or described in the present application and / or in the prior US Pat. (B) SRRS virus producing more than six sg mRNAs when cultured or when passaged by CRL 11171 cells, (c) RSVRSs which produce at least 40% of CRL 11171 cells. severe lung lesions or inflammatory lung consolidation in piglets born 5 weeks of age and delivered by cesarean section in piglets that were deprived of colostrum 10 days after infection, (d) a PRRSV isolate having a genome encoding a protein with the minimum homology to a VSRRS QLA, described in Table 2 below and / or (d) an isolate of any RSVRS having the identifying characteristics of one of these viruses. The vaccine of the present invention is effective in protecting a pig against infection caused by a respiratory and reproductive syndrome of swine (RSRF) virus. A vaccine protects a pig against RSVRS infection if, following administration of the vaccine to one or more uninfected pigs, a subsequent attack with a biologically pure virus isolate (e.g., VR 2385, VR 2386 or other virus isolates described below), result in a lesser severity of any large or histopathological changes (e.g., lung injury) and / or a decrease in disease symptoms when compared to changes or symptoms normally caused by isolated on similar swine which are not protected (ie, for adequate control). More particularly, it may be demonstrated that the vaccine of the present invention may be effective by administering the vaccine to one or more appropriate pigs in need thereof and, after a suitable period of time (e.g., 1-4 weeks), giving a reinforcement with a large sample (103 -7 TCID50) [thiocarbonyldiimidazole] from a biologically pure RSVAR isolate. After about one week, a blood sample is then withdrawn from the injected swine and a test is performed to try to isolate the virus from the blood sample (see, for example, the virus isolation process exemplified in experiment VIII presented at follow). Isolation of the virus is an indicator that the vaccine may be ineffective and failure to isolate the virus is an indicator that the vaccine may be effective.

Thus, the efficacy of the present vaccine can be quantitatively assessed (ie, a decrease in the percentage of consolidated lung tissue as compared to an appropriate control group) or qualitatively (e.g., isolation of the RSVRS from the blood, detection of an antigen from RSVRS in a lung, a tissue sample from a lymph node or tonsils by an immunoperoxidase assay procedure [described in detail below, etc.]. The symptoms of the reproductive and respiratory disease of the swine 13 may be evaluated quantitatively (e.g., by measuring the temperature), semi-quantitatively (e.g., the severity of the respiratory distress [explained in detail below] or qualitatively (e.g. the absence of one or more symptoms or the reduction of severity of one or more symptoms, such as cyanosis, pneumonia, cardiac and / or cerebral lesions, etc.)

An uninfected pig is a pig which has either not been exposed to an infectious agent of the reproductive and respiratory disease of the pigs or has been exposed to the infectious agent of the reproductive and respiratory disease of the pigs but does not manifest the symptoms of the disease. An affected pig is the one who manifests symptoms of SRRS or from which it was possible to isolate the RSRRS.

Clinical signs or symptoms of RSVRS may include lethargy, difficulty breathing, " a cavernous sound " (forced expiration), fevers, shivering, sneezing, coughing, edema in the eyes and occasionally conjunctivitis. Lesions may include extensive and / or microscopic lung lesions, myocarditis, lymphadenitis, encephalitis, and rhinitis. In addition, less virulent or non-virulent forms of RSVRS and of the Iowa strain have been found which may cause either a subset of the symptoms described above or even none of the symptoms. Nevertheless, less virulent or non-virulent forms of RSVRS may be used according to the present invention against the reproductive and respiratory syndrome of the swine. The phrase "polynucleic acid" refers to RNA or DNA, as well as to mRNA and cDNA, which corresponds to the complement of RNA or DNA isolated from the virus or infectious agent. A "QLA" refers to an open reading frame or to a segment encoding the polypeptide, isolated from a genome of a virus, including a genome of the RSVRS. In the polynucleic acid of the present invention, a QLA may be included, in part (as a fragment) or whole and may overlap with the 3 'or 5' sequence of an adjacent QLA (see, for example, Figure 1 or experiment 1 Next). A "polynucleotide" is equivalent to a polynucleic acid, but may define a distinct molecule or group of molecules (for example, as a subset of a group of polynucleic acids).

In the experiments described below in the present invention, the isolation, cloning and sequencing of BLAs 2 to 5 from (a) a low virulence EURVRS isolate and (b) two other EURVRS isolates were performed of variable virulence. The nucleotide and the deduced amino acid sequences of these three U.S. isolates were compared to corresponding sequences from other known RSRS isolates (see, for example, U.S. Serial No. 08 / 301,435). The results indicate that there are considerable genetic variations not only between the EU RSVR and the European RSVR, but also among the US isolates. The identity of the amino acid sequence among the seven isolates of the RSVR of the US studied was 91-99% at QLA 2, 86-98% in QLA 3, 92-99% in QLA 4, and 88-97% in QLA 5. The less virulent US isolate, known as VR 2431 (ISU 3927), has sequence variations higher in QLAs 2 to 4 than in QLAs 5 to 7 when compared to other isolates of the US viruses. Three hypervariable regions were identified whose antigenic potential was identified in the major envelope glycoprotein encoded by QLA 5.

Comparison between pairs of QLA sequences 2 to 7 and phylogenetic tree analysis indicate the existence of at least three groups of RSVRS variants (or minor genotypes) within the major genome of the RSVRS of the US 0 isolates of the US virus minus virulent form a distinct branch of the other isolates from known US viruses with different virulence. The results of this study have implications for the RSVRS taxonomy and the development of a vaccine.

In another experiment, sg mRNAs were characterized in the cells infected with RSRRS. The data showed that a set of six or seven mRNAs sg nested at the 3 'common end was formed in cells infected with different RSRSV isolates. However, unlike some coronavirus and alphavirus, the SRS rs ssRNAs are not packaged in the virion and only the RRS genome of the PRRSV was detected in the purified virions. Variations in the number of sg mRNAs were also observed among different PRRSV isolates with different virulence. Subsequent analyzes of the sequence of QLAs 2 to 7 of two isolates of U.S. viruses and their comparison with European VL revealed the heterogeneous nature of the mRNA junction sequences to the leader of the RSVRS.

As demonstrated in Experiment 2 below, a set of six or more sg mRNAs nested at the common 3 'end was formed in cells infected with different PRRSV isolates. The presence of a nested set of sg mRNAs further indicates that the U.S. RSVRS, such as the European isolate of Leystad virus (VL), belong to the recently proposed family of Arteriviridae including VLDH, VAE and VFHS. Northern blot analysis " with QLA-specific probes indicates that the structure of the 16 rsRNA sg of the RSVRS is polycistronic (has several cistrons) and that each of the sg mRNAs, except for the 7 sg mRNA, contains multiple QLAs. Therefore, the sequence of each sg mRNA is contained in the 3 'portion of the next major sg mRNA, and not all 5' ends of sg mRNAs overlap the sequences of smaller sg mRNAs. There is, however, no apparent correlation between the number of sg mRNAs and virus pneumovirulence. An additional species, sg 4-1 mRNA, was found to contain a small QLA (QLA 4-1) with a coding capacity of 45 amino acids at its 5 'end.

In the following experiment 2, the SRS rsRNA mRNAs were found not to be packaged in the virions. The fact that sg mRNAs are packaged in the virions may depend on one of the mRNAs sg containing a packaging signal. Since the ssRNA mRNAs of the RSVRS are not packaged in the virions, the RSVR encapsidation signal is probably located in the QLA 1 region, which is unique to the virus genome but is not present in mRNAs sg.

In Experiment 2 below, the junction segments (the leading mRNA junction segments) of the mRNAs sg 3 and 4 of two isolates of the U.S. isolates of the RSVRS, VR 2474 (ISU 79) and VR 2475 (ISU 1894). Knowledge of the sequence identities of the mRNA-junction sequence to the leader provides means to effectively produce (a) chimeric viruses for use as an infectious clone and / or as a vaccine and (b) vectors for inserting or 'carrying and bringing ( shuttling) one or more genes in an appropriate host capable of being infected. Methods for designing and producing such chimeric viruses, infectious clones, and vectors are known to those skilled in the art (see, for example, Sambrook et al., &Quot; Molecular Cloning: A Laboratory Manual ", 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The mRNA-junction sequence leading to the mRNAs sg 3 and 4 of two isolates are different (TTGACC for ISU 79 mRNA 4-1, GTAACC for mRNA 3 and TTCACC for mRNA 4). Most nucleotide differences in the junctions occur in the first 3 nucleotides. The last 3 nucleotides do not vary, suggesting that the junction of the leader sequence to sg mRNA bodies occurs at the 5 'end of the mRNA junction sequence to the leader. Similar observations were reported for VL, VAE and VDHL. The acquisition of the additional 4-1 sRNA mRNA in the VR 2474 isolate (ISU 79) is due to a single nucleotide substitution which gives rise to a new leading mRNA junction sequence. This substitution occurs at the last nucleotide of the junction segment, suggesting that the last nucleotide of the mRNA junction element at the leader is critical for leader binding and for the initiation of transcription.

Although sequence homology between the leader and the intergenic regions of coronaviruses leads to the hypothesis that base pairing may be essential in leader-initiated transcription, there is no experimental evidence that has proven the need for base pairing in transcription of mRNAs sg. For example, the sequence at the 3 'end of the leader that is involved in the fusion process in both coronaviruses and arteroviruses remains unknown. 18 Several lines of evidence support the leader-initiated transcription mechanism for coronaviruses, but the presence of negative helix sg mRNAs and subgenomic replicative intermediates (sg IR) in coronavirus-infected cells suggests that the mechanism involved in sg mRNA synthesis is more complex than a mere base pairing of the leader sequence with the join sequence. However, negative helix sg mRNAs were not detected in arteriviruses except for VDHL and sg IRs were only detected in VAE infected cells. For this reason, the synthesis of sg mRNAs in arteriviruses and, in particular, in RSVRS may be less complicated than in coronaviruses. Analysis of the QLA sequences 2 to 7 of the U.S. RSVRS isolates and comparison of the consequences with the VL shows the heterogeneity of the mRNA junction sequences to the leader. The presence of mRNA-junction elements to the leader at positions that do not correspond to a sg mR raises the question whether a small set of only six nucleotides that are conserved in the leader and junction sequences in the genomes of the RSRRS and other arteriviruses , is sufficient for efficient binding of the leader to these specific junction sites upstream of the QLAs. This apparent discrepancy can, however, be explained by the following two possibilities.

First, it is expected that additional structural elements, such as secondary structures or the sequences surrounding the mRNA-binding junction to the leader, are involved in the fusion (binding) of the leader to the specific sites. In the VHR, the sequence flanking the consensus sequence (mRNA junction sequences to leader) of UCUAAAC affects the efficiency of sg D1 RNA transcription and that the consensus sequence was necessary but not sufficient, for alone, for D1 mRNA synthesis.

Secondly, the distance between the two junction regions of the mRNA to the leader can affect the transcription of mRNAs sg. The mRNA-junction region at the downstream leader was shown to be decreasing the synthesis of the RNA sg D1 RNA from the mRNA junction region to the upstream leader. This deletion was significant when the separation of the two mRNA junction sequences to the leader was less than 35 nucleotides. However, no significant inhibition of major sg D1 RNA synthesis (from the upstream leading mRNA junction sequence) was observed when the two regions of the mRNA-leader junction were separated by more than 100 nucleotides.

The experimental results presented above are consistent with the observations reported in experiment 2 below, in which an additional species of sg 4-1 mRNA, in addition to sg4 mRNA, is observed in some of the isolates of the RSVRSS. The mRNA leader mRNA sg 4 and 4-1 junction sequences of the VSRRSS Iowa strains are separated by about 226 nucleotides. Therefore, the synthesis of the major 4-1 mRNA mRNA of the upstream mRNA-mating junction sequence was not suppressed by the presence of the downstream leader mRNA binding sequence.

We have found, on the other hand, multiple potential mRNA-to-leader junction sequences at different positions upstream of QLA 3, 5, 6 and 7, but in the " Northern blot " no sg mRNA corresponding to these mRNA junction elements to the leader was found. Most of these mRNA-to-leader junction sequences are separated by less than 50 nucleotides from the mRNA junction region to the downstream leader, except for QLA 7 (where the two potential leader mRNA junction sequences are separated by 114 nucleotides). However, sg 7 mRNA in the " Northern blot " appears as a widely diffused band. Therefore, transcription of the major mRNA mRNA from the mRNA junction sequence to the upstream leader may thus not be significantly suppressed by the downstream junction sequence, but this can not be easily differentiated by " Northern " blot "of the abundant sg 7 mRNA.

The QLA's 2-7 of the ISU-12 RSV isolate (filed October 30, 1992, American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, EU, under accession numbers VR 2385 [ plaque] and VR 2386 [purified non-plate] and QLA's 6-7 of the ISSR-22 isolates ISU-55, ISU-3927 (filed September 29, 1993 at the American Type Culture Collection, VR 2429, VR 2430 and VR 2431, respectively), isolates ISU-79 and ISU-1894 (filed August 31, 1994, American Type Culture Collection under accession numbers VR 2474 and VR 2475, respectively) are described However, the techniques used to isolate, clone and sequence these genes can also be applied for the isolation, cloning and sequencing of polynucleic acids from the genome of any one of the genomes of the genome. VSRRS.

For example, primers to produce relatively large amounts of DNA by polymerase chain reaction (and, if desired, to produce RNA by transcription and / or protein by translation according to known in vivo or in vitro methods) can be designed (for example, QLA's 2-7 of VR 2385, VR 2429, VR 2430, VR 2431, VR 2474, VR 2474, VR 2475 (ISU-1894)) and the sequence information was determined when more than one sequence was determined from the genome of the VSRRSS , VR 2332 and the Lelystad virus). A region of about 15 to 50 nucleotides in length, having at least 80% and preferably 90% identity, is selected from the given sequences. A region where deletion occurs in one of the sequences (for example, at least 5 nucleotides) may be used as the base in the preparation of an initiator for the selective amplification of the polynucleic acid of one strain or type of the RSVPR over the others , for the differential diagnosis of the RSVRS strains of the United States and Europe)

Once the polynucleic acid is amplified and cloned into an appropriate host by known methods, the clones can be screened with a probe designed based on the sequence information described in the present invention. For example, a region of about 50 to about 500 nucleotides in length is chosen based on either a high degree of identity (e.g., at least 90%) between two or more sequences (for example, in QLA's 6-7 the Iowa RSVRS strains described below in Experiment III) and a polynucleotide of suitable length and sequence identity may be prepared by known procedures (such as the automatic synthesis or restriction of a suitable fragment of a polynucleic acid containing the region PCR amplification using primers that specifically hybridize to the polynucleotide and isolation by electrophoresis). The polynucleotide may be labeled, for example, with 32P (for radiometric identification) or labeled with biotin (for detection by fluorimetry). The probe is then hybridized with the polynucleic acids of the clones and detected by known methods.

Applicants have discovered that one more of the 2-4 ABQs may be related to the virulence of the RSVRS. For example, at least one isolate of the RSVAR having relatively low virulence also appears to have a deletion in QLA 4 (see, for example, experiments VIII-XI in U.S. Serial No. 08 / 301,435 ). In addition, the known less virulent isolate (VR 2431) exhibits a relatively high degree of variance in information on the sequences of both the nucleotides and the amino acids in ASFs 2-4, as compared to other isolates of the U.S.

In one embodiment, the invention relates to a polynucleic acid obtained from a PRRSV isolate, which confers, directly or indirectly, immunogenic protection against subsequent enhancement with a PRRSV, but wherein the polynucleic acid is deleted or mutated to such an extent that makes the RSVRS, which contains the polynucleic acid, in a phenotype with low virulence (i.e., a "low virulence" (vb) (see corresponding explanation in U.S. Serial No. 08 / 301,435) or (also referred to as " deletion mutant "). Preferably, one or more of the ABGs 2-4 is / are deleted or mutated to the point of transforming the SRRS virus to non-virulent. , it may be desirable to maintain regions of one or more QLAs 2-4 in the present polynucleic acid which (i) encode an antigenic peptide fragment and / or an immunoprotector and (ii) do not confer virulence to the SRRS virus containing the acid poly The present invention also encompasses a PRRSV per se wherein one or more of the ABRs 2-4 has been deleted or mutated to a point which renders it either less virulent or non-virulent. Such a virus is useful as a vaccine or as a vector that will serve to transform a suitable host (e.g., MA-104 cells, PSP 36, CRL 11171, MARC-145 or porcine alveolar macrophage cells) with a heterologous gene. Preferred heterologous genes, which may be expressed using the elimination mutant of the present invention, may include those encoding a protein or an antigen in addition to the swine reproductive and respiratory syndrome virus gene (eg, pseudorabies and / or swine influenza virus and / or antigens containing polypeptides, a porcine growth hormone, etc.) or a polypeptide-based adjuvant (as set forth in U.S. Serial No. 08 / 301,435 for the composition of the vaccine).

It may also be desirable in certain embodiments that the polynucleic acid of the present invention contains, for example, the 3 'end region of a RSVRS (e.g., 200 to 700 nucleotides in length), wherein at least part of it may overlap with the 5 'region of the LFA immediately downstream. Also, when the 3 'end region of a LFA can overlap the 5' end region of the LFA immediately downstream, it may be desirable to maintain the 5 'end region of the LFA that overlaps the LFA immediately downstream.

Applicants have also found that QLA 5 in the genome of the RSVRSS appears to be related to virus replication in host mammalian cells capable of maintaining a culture while infected by RSVRS. Accordingly, the present invention also relates to polynucleic acids obtained from a PRRSV genome in which PRA 5 may be present in multiple copies (referred to as "overproduction mutant." For example, the present polynucleic acid may contain at least less two, and most preferably, 2 to 10 copies of QLA 5 from a high replication (er) phenotype of the RSVRS isolate.

Interestingly, the VRSRV isolate VR 2385 (ISU-12) has a surprisingly high number of potential initiation codons (ATG / AUG sequences) near the 5 'end of QLA 5, possibly indicating alternative initiation sites for this gene. There may thus exist alternative forms of the protein encoded by the QLA 5 of a PRRSV isolate, in particular when the alternative QLA's encode a protein having a molecular weight similar to that determined experimentally (e.g., from about 150 to about 250 amino acids in length). The most likely coding region for VLA 2385 QLA 5 (ISU-12) is shown in figure 1.

Deleted and overproduced mutants can be produced according to known methods. For example, a mutant polynucleic acid may be prepared which contains a "silent" alteration or a degenerate change in the sequence of a region encoding a polypeptide. In selecting and producing an appropriate degenerative mutation, a known polynucleic acid sequence can be substituted for a known restriction enzyme (see, for example, Experiment 2). Thus, if a silent, degenerate mutation is produced at one or both of the 3 'ends of a LFA and at the 5' end of a LFA at 25 downstream, a synthetic polynucleic acid (called a 'cassette') may also be inserted. contain a polynucleic acid encoding one or multiple copies of a QLA 5 protein product of er, a RSVR or other envelope protein of the virus and / or an antigenic fragment of a PRRSV protein. The 'cassette' may be preceded by a suitable initiation codon (ATG) and may be appropriately terminated with a 3 'end termination codon (TAA, TAG or TGA). It is clear that an oligonucleotide sequence which does not encode a polypeptide can be inserted or, alternatively, no cassette may be inserted. In this case, the so-called elimination mutant can be provided. The isolated and / or purified polypeptide of the present invention may be one or more encoded by a "low virulence mutation" of one or more of VLA-2430 (ISU-55) QLAs 2, 3, and 4 (or a non-virulent fragment having at least 5 amino acids in length) wherein one or more of the 12-14 positions of the polypeptide encoded by QLA 2 are RGV (wherein " R " (arg), " G " " V " represent the abbreviation of a letter of the corresponding amino acids), positions 44-46 are LPA, position 88 is A, position 92 is R, position 141 is G, position 183 is H, position 218 is S, position 240 is S and positions 252-256 are PSSSW or any of combinations thereof. Other identities of amino acid residues which may be further combined with one or more amino acid position identities referred to above include those of heading 174 (I) and heading 235 (M).

The purified isolate and / or polypeptide of the present invention may also be encoded by a VLA 26 2430 QLA 3 (ISU-55), wherein one or more of the amino acid identities specified may be selected from those in positions 11 ( L), 23 (V), 26-28 (TDA), 65-66 (QI), 70 (N), 79 (N), 93 (T), 100-102 (KEV), 134 (K), 140 (N), 223-227 (RQRIS), 234 (A) and 235 (M) or any combination thereof, which may then be combined with one or more of the positions 32 (F), 38 (M), 96 P), 143 (L), 213-217 (FATS), 231 (R), and 252 (A). The isolated and / or purified polypeptide of the present invention may further be encoded by VLA 2430 (ISU-55) QLA 4 wherein one or more of the amino acid identities specified may be selected from those in positions 13 (E), (I) or any of combinations thereof, which may then be combined with one or more of the positions 2-3 (A) , 51 (G) and 63 (P). Preferably, the nucleic acid of the present invention encodes at least one antigenic region of the SRRSV envelope protein. More preferably, the polynucleic acid of the present invention encodes a hypervariable region of the product of a PRRSV QLA 5 protein (see discussion below) or (b) contains at least one copy of the QLA-5 gene of an isolate with a phenotype of high virulence (ev) (see the description of the " ev phenotype " in U.S. Serial No. 08 / 301,435) and a sufficiently long fragment, region or sequence of at least one of QLA-2, QLA-3, QLA-4, QLA-5 and / or QLA-6 from the genome of a PRRSV isolate to encode an antigenic region of the corresponding protein (s) protecting against a subsequent enhancement with, for example, a VSRRS isolate with ev. The polynucleic acid of the present invention may contain at least one entire envelope protein encoded by QLA-2, QLA-3 and QLA-5 of a RSVRS, and the polynucleic acid of the present invention excludes or modifies a sufficiently long portion of one of the QLAs 2-4 of a RSVR to render the RSVRS, which contains it, either low virulent or non virulent.

A further embodiment of the present invention includes a polynucleotide encoding an amino acid sequence of a hypervariable region of the RSVRS QLA 5. Thus, such polynucleotides encode one (or more) of the following amino acid sequences: TABLE 1

Hypervariable region 2 (positions57-66)

Hypervariable Region 1 (positions 32-38)

Hypervariable Region 1 (positions 120-128)

LICFVIRLA LTCFVIRFA LICFVIRFT LACFVIRFA LTCFVIRFV LTCFIIRFA FICFVIRFA FVCFVIRAA NGNSGSN SNDSSSH SSSNSSH SANSSSH HSNSSSH SNSSSSH NNSSSSH NGGDSST (Y)

ANKFDWAVET ANKFDWAVEP AGEFDWAVET ADKFDWAVEP ADRFDWAVEP SSHFGWAVET

In this embodiment, the polynucleotide may further encode other amino acid sequences of the RSVRS QLA 5 (as depicted in Figure 3 or in U.S. Serial No. 08 / 301,435), provided that one or more of the hypervariable regions at positions 32-38, 57-66 and / or 120-128. (The present invention specifically excludes the proteins and polynucleotides of VLA 5 and VR 2332.) The present invention also relates to a purified preparation which may comprise, consisting essentially of or consisting of a polynucleic acid, a vector or in a plasmid having a sequence of formula V: wherein ε, which is optionally present, represents a sequence of the 5 'end polynucleotide which provides a means for the operational expression of α, β, γ and δ polynucleotides; ζ represents a polynucleotide of the formula KTVACC, wherein K represents T, G or U, and V represents A, G or C '; i represents a maximum polynucleotide having about 130 nucleotides (preferably at most 100) in length; κ represents a polynucleotide which comprises one or more genes selected from the group consisting of a conventional marker or an α, β, γ reporter gene and their operatively linked combinations, wherein α encodes at least one of said polypeptides and β is at least one copy of a QLA 5 from an Iowa strain of RSVRS or an antigenic fragment thereof (e.g., one or more hypervariable regions), preferably a full length copy of a high replication (er) phenotype; and γ encodes at least one polypeptide or an antigenic fragment encoded by a polynucleotide selected from the group consisting of QLA 6 and QLA7 of an Iowa strain of the RSRRS and regions thereof encoding the antigenic fragments; and ξ, which is optionally present, represents a 3 'end polynucleotide sequence that does not suppress the operational expression of α, β, γ and δ polynucleotides which may be operably linked to ε (e.g., in a plasmid), wherein the polynucleic acid is not comprised of the sequence set forth in SEQ ID NO: 13 of WO 96/016619.

Suitable markers or reporter genes include, for example, those that confer resistance to an antibiotic such as neomycin, erythromycin or chloramphenicol; those encoding a known detectable enzyme, such as β-lactamase, dihydrofolate reductase (DHFR), horseradish peroxidase, glucose-6-phosphate dehydrogenase, alkaline phosphatase and the enzymes described in U.S. Patent No. No. 4,190,496, col. 32, line 33 to col. 38, line 44 (incorporated herein by reference), etc .; and those encoding a known antibody (e.g., murganhoo IgG, rabbit IgG, mouse IgG, etc.) or antigenic proteins known as protein A, protein C, bovine serum albumin (BSA), hemiocyanin from (KLH), bovine gamma globulin (GBG), lacto-albumin, polylysine, polyglutamate, lectin, etc. The polynucleotide is preferably a polynucleotide having a sequence with at least 80% homology to a polynucleotide sequence of a RSVRV genome, located between the sequence of the mRNA binding to the leader and the initiation codon of the QLA immediately downstream. 'About 130 nucleotides in length refers to a length of a polynucleotide that does not adversely affect the operative expression of k. For example, in ISU 79, a leader mRNA junction sequence that does not suppress the expression of QLA 7,129 bases upstream of the QLA 7 start codon (see experiment 2 below) can be found. Examples of suitable sequences for the L polynucleotide can be deduced from the sequences shown in Figures 1 and 9. The polynucleic acid of the present invention may also comprise, consisting essentially of or consisting of combinations of the above sequences, either as a mixture of polynucleotides or ligated by covalence in a head-to-tail (parallel or anti-parallel) or head-to-head connection. Complementary polynucleic acids for the above-mentioned sequences and for their combinations (anti-parallel polynucleic acid) are also encompassed in the present invention. Thus, in addition to having multiple copies or variants of QLA 5, the polynucleic acid of the present invention may also contain multiple copies or copies of variants of one or more of the QLAs 1-7, including antigenic or hypervariable regions of the QLA 5 strain Iowa of the RSVRS.

A library of recombinant clones (for example, using E. coli as a host) may be prepared, containing restriction fragments appropriately prepared from a PRRSV genome (for example, inserted into a suitable plasmid that can be expressed in the host ), in a manner similar to the processes described above, to the experiments described below and in U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435. The clones are then screened with a suitable probe (for example, based on a conserved sequence of the BLAs 2-3; see, for example, Figure 22 of U.S. Application Serial No. 08 / 301,435). Positive clones may be selected and grown to an appropriate level. The polynucleic acids can then be isolated from the positive clones according to known methods. Appropriate primers for PCR, as described above, may be designed and prepared to amplify the desired region of the polynucleic acid. The amplified polynucleic acid can then be isolated and sequenced by known methods.

In the context of the present application, "homology" refers to the percentage of identical nucleotides or amino acid residues in the sequences of two or more viruses, aligned according to a standard homology determination method (for example, computer programs MACVECTOR or GENEWORKS, aligned according to the procedure described in Experiment III of U.S. Application Serial No. 08 / 301,435).

Preferably, the isolated polynucleic acid of the present invention encodes a protein, a polypeptide or an antigenic fragment thereof that is at least 10 amino acids in length and wherein non-homologous amino acids which are not essential for antigenicity can be conservatively substituted . An amino acid residue can be conservatively substituted in a protein, a polypeptide or an antigenic fragment thereof if it is replaced by an element of its polarity group as defined below:

Amino Acids: Aspartic Acid (Asp), Glutamic Acid (Glu), Asparagine (Asn), Glutamine (Gin) 32 Amino Acids: Lysine (Lys), Arginine (Arg), Histidine

Amino nonionic, hydrophilic acids: serine (Ser), threonine (Tre), cysteine (Cis), asparagine (Asn), glutamine (Gin)

Amino acids containing sulfur: cysteine (Cys), methionine (Met)

Aromatic, hydrophobic amino acids: phenylalanine (Phen), tyrosine (Tyr), tryptophan (Trp) Non-aromatic, hydrophobic amino acids: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro)

More particularly, the polynucleic acid of the present invention encodes one or more of the proteins encoded by the second, third, fourth, or fifth open reading frames (SLFs 2-5) of the PRRSV isolate.

QLAs 6 and 7 are probably not candidates to control virulence and replication phenotypes of the RSRRS, since the nucleotide sequences of these genes are highly conserved between the highly virulent (ev) and the low virulence (bv ) (see Experiment III of U.S. Application Serial No. 08 / 301,435). However, QLA 5 in the RSVRV isolates appears to be less conserved between high replication (er) and low replication (br) isolates. Therefore, it is believed that the presence of a QLA 5 of a rsRVR isolate of er in the polynucleic acid of the present invention will stimulate the production and expression of a recombinant vaccine produced from polynucleic acid.

In addition, VSRRS QLA 5 contains three hypervariable, hydrophilic regions, normally associated with antigenicity in a polypeptide.

It is preferred that the polynucleic acid of the present invention, when used for immunoprotective purposes (for example, in the preparation of a vaccine), contains at least one copy of QLA 5 of VR 2430 (ISU-55) (i.e., an isolate that grows to a title of 106-107 TCID50 in, for example, CRL 11171 cells; see also the discussions in Experiments VIII-XI of U.S. Application Serial No. 08 / 301,435).

On the other hand, the bv VR 2431 isolate appears to be an elimination mutant, relative to the Vβ isolates (see experiment III and VIII-XI of U.S. Application Serial No. 08 / 301,435). The elimination appears to be in QLA 4, given the " Northern blot " analysis. Accordingly, when used for immunoprotective purposes the polynucleic acid of the present invention preferably does not contain a region of the QLA 4 of a virus isolate responsible for the high virulence; and most preferably excludes the QLA region 4 which does not overlap with the adjacent QLAs 3 and 5.

It is also known (at least for RSVSR) that neither the nucleocapsid protein nor its antibodies give pigs immune protection against RSVRS from swine. Accordingly, the polynucleic acid of the present invention, when used for immunoprotective purposes, may contain one or more copies of one or more regions of QLAs 34 2, 3, 4, 5 and 6 of a PRRSV isolate encoding a region of the virus envelope protein but does not produce the symptoms or histopathological changes associated with RSVR when administered to the pigs. Preferably, this region has, from the immunological point of view, cross-reactivity with the antibodies to the envelope proteins of other PRRSV isolates.

Likewise, the protein encoded by the polynucleic acid of the present invention provides protection against SRRS to a pig which has been administered a composition comprising the protein and the antibodies to this protein have, immunologically, cross-reactivity with the envelope proteins from other isolates of the RSVRS.

Relatively short (about 20 bp or greater) polynucleic acid segments may be used in the genome of a virus to screen for or identify tissue and / or biological fluid samples from infected animals and / or to identify related viruses by processes described in the present invention and known to those skilled in the art of veterinary medicine and the diagnosis of viruses and veterinary medicine.

A further embodiment of the present invention relates to one or more proteins or antigenic fragments thereof of VR 2430 (ISU-55).

As described above, an antigenic fragment of a SRRS virus protein is at least 5 amino acids in length, particularly and preferably at least 10 amino acids in length, and provides or stimulates an immunological protection response in a pig which has been administered a composition containing the antigen fragment.

Methods of determining the antigen portion of a protein are known to those skilled in the art (see previous description). In addition, it is also possible to determine an essential antigenic fragment of a protein by first showing that the full length protein is antigenic in a host animal (e.g., a pig). If the protein remains antigenic in the presence of an antibody that specifically binds to a particular region or protein sequence, then that region or sequence may not be essential for immunoprotective. On the other hand, if the protein is no longer antigenic in the presence of an antibody that specifically binds to a particular region or protein sequence, then that region or sequence is considered essential for antigenicity.

The three hypervariable regions in the RSVAR ALFA 5 were identified by comparing the amino acid sequences of the ALLA product of all available RSVRS isolates (see, for example, Figure 2D). The amino acid variations in these three regions are significant and are not structurally conserved (Figure 2D). All three hypervariable regions are hydrophilic and antigenic. Thus, these three regions are likely to be exposed to the virus membrane and to be under the pressure of selection of the host immune system. Hypervariable regions are the antigenic determinants in the envelope protein of QLA 5. The present invention also relates to a protein or antigenic fragment encoded by one or more of the polynucleic acids defined above. The antigenic proteins and fragments of the present invention are useful for immunizing pigs against RSVRS, for serological tests to screen pigs exposed to RSVRS infection (in particular to the RSVRS strain of Iowa), etc. The protein of the present invention may be selected from the group consisting of proteins encoded by QOLs 2-5, ISU-55 (VR 2430); antigenic regions of at least one of these proteins having a length between 5 amino acids to less than the total length of the protein;

Preferably, the protein of the present invention has a sequence encoded by a QLA selected from the group consisting of VA 2430 ALFAs 2-5 (see, for example, Figure 2A-D); their variants which provide effective immunological protection to a pig to which variants in which there are from 1 to 100 (preferably 1 to 50 and most preferably 1 to 25) deletions in the amino acid sequence; and with their antigenic fragments, at least 5 and preferably 10 amino acids in length, which provide effective immunological protection to a pig to which the fragments have been administered.

More preferably, the protein variant or protein fragment of the present invention has a binding affinity (or association constant) of at least 1% and preferably at least 10% of the binding affinity of the complete, naturally occurring protein a monoclonal antibody that specifically binds to the naturally occurring full length protein (i.e., the protein encoded by the RSVRS QLA). The present invention also relates to a method of producing a polypeptide comprising the expression of the polynucleic acid of the present invention in an operative expression system and the purification of the expressed polypeptide obtained from the expression systems. Suitable expression systems include those conventionally used for both in vitro and in vivo expression of proteins and polypeptides, as an in vitro rabbit reticulocyte expression system, and for in vivo expression of a modified or chimeric RSVP (used to infect a host cell line with the ability to be infected, such as MA-104, CAL 11171, PSP-36, PSP-36-SAH, MARC-145 and alveolar pig macrophages) or a conventional expression vector containing the acid (e.g., a thymidine kinase promoter, SV40, etc.) to be used in conventional expression systems (for example, bacterial plasmids and corresponding host bacteria, expression in yeasts and the corresponding host yeasts, etc.). The expressed polypeptide or protein is then purified or isolated from the expression system by conventional purification and / or isolation procedures.

Other features of the present invention which will appear in the descriptions of the exemplary embodiments are given for illustration of the present invention, but are not limitative thereof. EXPERIENCE 1

Summary:

Sequences 2 to 5 of a U.S. RSVRS isolate with low virulence, "moderate" virulence, and high virulence were determined and analyzed. Comparisons made with other known sequences from other SRSRV isolates showed that there are considerable sequence variations, both at the nucleotide and amino acid levels, in FDSs 2 to 5 of seven isolates of the U.S. RSVR with different virulence. However, QLAs 6 and 7 of these seven American isolates are highly conserved (U.S. Serial No. 08 / 301,435). Large variations in QLAs 2 to 7 were also found between European VL and U.S. isolates. The less virulent U.S. isolate known, VR 2431 (ISU-3927), showed the greatest sequence variation, compared to other U.S. isolates.

The phylogenetic relationship of the isolates from the US was also analyzed. Phylogenetic analysis of US isolates 2 to 7 from isolates indicates that there are at least three groups of RSVRS (or minor genotype) variants within the main genotype of the US RSVR. Accordingly, it is quite likely that more main or secondary genotypes will be identified as more isolated viruses are examined from different geographic regions.

Interestingly, the less virulent US isolate, VR 2431 (ISU-3927), forms a distinct branch from the other isolates in the US. Analysis of nucleotide and amino acid sequences also shows that VR 2431 isolate (ISU 3927) greater number of variations in QLAs 2 to 4 compared to other isolates in the US Many of these variations of isolate VR 2431 (ISU 3927) result in non-conserved amino acid substitutions. However, these non-conserved changes in isolate VR 2431 (ISU 3927), when compared to that of 39 other U.S. isolates, do not appear to be confined to a particular region; they are present in all QLAs 2 to 4. Therefore, a specific correlation between sequence variations and virulence of viruses is not fully justified (although some positions in QLA 3 appear to be possibly related to virulence, see Figure 2B, amino acids, which are in one or more of these positions, can serve as the basis for the mutation of other known proteins encoded by an ARVS QLA 3).

RESULTS: The identity of the amino acid sequences among the seven isolates of the RSVRS of the EU was 91-99% in ALF 2, 86-98% in ALF 3, 92-99% in ALF 4 and 88-97% in QLA 5. The less virulent known US isolate has the higher sequence variations in QLAs 2 to 4 than in QLAs 5 to 7, as compared to other isolates in the US Three hypervariable regions with antigenic potential were identified in the major glycoprotein envelope. The comparison between pairs of QLA sequences 2 to 7 and phylogenetic tree analysis suggest the existence of at least three groups of RSVRS (or minor genotype) variants within the major genome of the U.S. RSRRS isolated from the less virulent US, forms a distinct branch of the other isolates of the US with different virulences. The results of this study have implications for the RSVRS taxonomy and vaccine development. Figure 1 shows a comparison of the nucleotide sequences of QLAs 2 to 5 of the U.S. isolates VR 2431 (ISU 3927), VR 2429 (ISU 22) and VR 2430 (ISU 55) with other known RSRS isolates. The nucleotide sequence of VR 2385 (ISU 12) is shown at the top and only the differences are indicated. The start codon of each QLA is indicated with the + > and the stop codon of each QLA is indicated with an asterisk (*). The leader mRNA junction sequences for subgenomic mRNAs 3, 4 and 4-1 are underlined and the locations of the primer sequences relative to the initiation codon of each QLA are indicated by the minus sign (-) followed by the number of nucleotides upstream of each QLA. The sequences of VR 2385 (U.S. Patent Applications Serial Nos. 08 / 131,625 and 08 / 301,435), VR 2332, VR 2474 (ISU 79) and VR 2475 (ISU 1894) U.S. Serial No. 08 / 301,435) used in this alignment were previously reported. MATERIALS AND PROCESSES: Cells and viruses:

Cell line CRL 11171 (ATCC) was used to propagate the RSVRS. Cells were grown in Dulbecco's minimal essential medium (MEMD) supplemented with 10% fetal bovine serum (SBF) and 1X antibiotics (penicillin G 10,000 unit / ml, 10,000 mg / ml streptomycin and 25 mg / ml amphotericin B).

The three isolates of the RSVRS from the US used in this study, designated VR 2429 (ISU 22), VR 2430 (ISU 55) and VR 2431 (ISU 3927) from pig lungs obtained from different farms in Iowa during the eruption of the SRRS. The three isolates were purified three times on plaque in CRL 11171 cells before further experiments. Comparative studies of pathogenicity have shown that ISU 3927 isolate is the least virulent isolate among the 10 different isolates of the RSVRS of the EU. The isolate VR 2429 (ISU 22) is the most virulent isolate and the isolate VR 2430 (ISU 55) is "moderately »Pathogenic. All three virus isolates used in this experiment were up to seventh.

Isolation of Intracellular RRS Viruses:

Confluent monolayers of CRL 11171 cells were infected with three isolates of the EU RSVRS, VR 2429 (ISU 22), VR 2430 (ISU 55) and VR2431 (ISU 3927), respectively, with a multiplicity of infection (mdi) of 0.1. 24 hours after infection, the infected cells were washed three times with cold FCF buffer. Total intracellular RNAs were then isolated by extraction with guanidinium isothiocyanate and phenol-chloroform (Stratagene). The presence of the virus-specific RNA species in an RNA preparation was confirmed by " Northern blot " (data not shown). Total intracellular RNAs were quantified by spectrophotometry.

Reverse Transcription and Polymerase Chain Reaction (TI-RCP):

First, complementary DNA helices (c) were synthesized from total intracellular RNAs by reverse transcription using random primers as previously described (Meng et al., 1993, J. Vet. Diagn. Invest., 5: 254- 258). For the amplification of the entire protein coding regions of QLAs 2 to 5 42 of the three PRRSV isolates, two sets of primers were designed based on the VR 2385 and VL sequences. The primers JM259 (5'-GGGGATCCTTT GTGGAGCCGT-3 ') and JM260 (5'-GGGGAATTCGGGATAGGGAATGTG-3') amplified the sequences of QLAs 4 and 5 and primers XM992 (5'-GGGGGATCCTGTTGG-TAATAG (A) GTCTG-31 and XM993 (5'-GGTGAATTCGTTTTATTTCCCTCCGGGC-3 ') amplified the sequences of QLAs 2 and 3. The unique restriction sites (EcoRI or BamHI) were introduced at the 5' end of these primers to facilitate cloning. A degenerate base, G (A), was synthesized on primer XM992 based on the sequences of VR 2385 and LV (Meulenberg et al., 1993; U.S. Serial No. 08 / 301,435). PCR was performed as previously described (Meng et al., 1993, J. Vet. Diagn. Invest., 5: 254-258).

Cloning and nucleotide sequencing:

The PCR-TI products were analyzed by 0.8% agarose gel electrophoresis. The two fragments resulting from PCR, which represent QLAs 2 and 3, as well as QLAs 4 and 5, respectively, were purified by " glassmilk " (GENECLEAN kit, BIO 101, Inc.). Each of the purified fragments was digested with BamHI and EcoRI and cloned into the pSK + vector as described previously (Meng et al., 1993). E. coli DH 5a cells were used for transformation of the recombinant plasmids. The white colonies were selected and grown in an LB broth containing 100 mg / mL ampicillin. Lysis of plasmid-containing E. coli cells using the Qiagen column (QIAGEN Inc.) was done with lysozyme. 43

The plasmids containing the viral inserts were sequenced with an automated DNA sequencer (Applied Biosystem, Inc.). Three or more independent cDNA clones were sequenced representing the complete sequence of the BLAs 2 to 5 of each of the three isolated from the PRRSV with universal and inverse primers. Several virus-specific primers, XM969 (5'-GATAGAGTCTGCCCTTAG-3 '), XM970 (5'-GGTTTCACCTAGAATGGC-3'), XM1006 (5'-GCTTCTGAGATGAGTGA-3 '), XM077 (5'-CAACCAGGCGTAAACACT- 3 ') and XM078 (5'-C TGAGCAAT TACAGAAG-3'), to determine the sequence of QLAs 2 to 5.

Sequence analysis:

Sequence data were combined and analyzed using MacVector (International Biotechnologies, Inc.) and GeneWorks (IntelliGenetics, Inc.) computer software programs. The phylogenetic analyzes were performed using the software package PAUP, version 3.1.1 (David L. Swofford, Illinois Natural History Survey, Champaign, IL). The PAUP application uses an algorithm with the maximum parsimony criterion to construct the phylogenetic tree. RESULTS:

Analyzes of the nucleotide sequences of QLAs 2 to 5:

The sequences of the BLAs 2 to 5 of five isolates of the RSVRS, VR 2474 (ISU 79), VR 2475 (ISU 1894), VR 2429 (ISU 22), VR 2430 (ISU 55) and VR 2431 (ISU 3927) and compared with other known RSRS isolates including VR 2385, VR 2332 and VL (Meulenberg et al., 1993). The sequences of QLAs 6 and 7 from VR 2385, 44 VR 2429 (ISU 22), VR 2430 (ISU 55), VR 2474 (ISU 79), VR 2475 (ISU 1894) and VR 2431 (ISU 3927) isolates were reported previously (U.S. Application Serial No. 08 / 301,435). Isolates used in this experiment have already been shown to differ in pneumovirulence in experimentally infected swine (U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435). VR 2431 (ISU 3927) is the least virulent isolate among the ten different U.S. RSVRS isolates (U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435).

As in other U.S. RSVR isolates, QLAs 2 to 4 of these isolates overlap each other (Figure 1). However, unlike in VL, QLAs 4 and 5 of U.S. isolates are separated by 10 nucleotides (Figure 1). The VLA QLAs 4 and 5 are overlapped by one nucleotide. Replacement of a single A nucleotide of the initiation codon of QLA 5 in VL to T in the EU isolates places the QLA 5 initiation codon of the 10 nucleotide isolates downstream of the QLA 4 termination codon. a non-coding sequence of 10 nucleotides appears between the 4 and 5 of isolated isolates from the known US (Figure 1).

VLA 2474 QLA 2 (ISU 79) is 3 nucleotides shorter than other U.S. isolates. Replacement of a single TGG nucleotide by TAG immediately prior to the QLA 2 termination codon creates a new VR termination codon 2474 (ISU 79) (Fig. 1). A 3 nucleotide deletion was also found in QLA 5 of VR 2431 (ISU 3927), and compared to other U.S. isolates (Figure 1). The size of the ALL 2 to 5 ALL of all isolates from the US is identical, except for VLA 2473 QLA 2 (ISU 79) and QLA 45 5 VR 2331 (ISU 3927), which are both 3 nucleotides shorter than other QLAs (Fiqura 1).

Comparisons of the QLA sequences 2 to 5 of the seven U.S. RSRS RSV isolates shown in Figure 1 indicate that there are considerable variations of nucleotide sequences in QLAs 2 to 5 of the U.S. isolates (Figure 1). The identity of the nucleotide sequence was 96-98% in QLA 2, 92-98% in QLA 3, 92-99% in QLA 4 and 90-98% in QLA 5 between VR 2385, VR 2332, VR 2429 (ISU 22), VR 2430 (ISU 55), VR 2474 (ISU 79), and VR 2475 (ISU 1894) (Table 2). The less virulent isolate VR 2431 (ISU 3927) has the highest number of variations among the seven U.S. RSVRS isolates (Figure 1 and Table 2). The identity of the nucleotide sequence between VR 2431 (ISU 3927) and other isolates from the EU was 93-94% in QLA 2, 89-90% in QLA 3 and 91-93% in QLA 4 (table 2) . Like QLAs 6 and 7 (U.S. Serial No. 08 / 301,435), QLA 5 of VR 2431 (ISU 3927) have no significant changes for the elimination of 3 nucleotides (Figure 1). The QLA 5 of VR 2431 (ISU 3927) shares 91-93% of the nucleotide sequence identity to the QLA 5 of other U.S. isolates (Table 2).

However, large sequence variations were found in QLAs 2 to 5 between VL and U.S. isolates (Figure 1 and Table 2). The identity of the nucleotide sequence between the VL and the US isolates was 65-67% in QLA 2, 61-64% in QLA 3, 63-66% in QLA 4 and 61-63% in QLA 5 (table 2). There are large genetic variations in QLAs 6 and 7 between the VL and the U.S. RSVRS (U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435). These results indicate that the less virulent isolate, VR 2431 (ISU 3927), is also less related to isolates from the US, with genetic variations occurring mainly in QLAs 2 to 4. Replacement of single TGG nucleotide by GAG before The codon termination codon in VLA 2 observed in VR 2474 (ISU 79) was also present in the isolate VR 2430 (ISU 55) and VR 2431 (ISU 3927), both of which produce seven sg mRNAs, but not in VR 2429 isolates ( ISU 22), VR 2475 (ISU 1894) or VR 2385, each synthesizing only six sg mRNAs (U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435). The results indicate that the 4-1 mRNA binding sequence to the leader of VR 2430 (ISU 55) and VR 2431 (ISU 3927) is most likely the same as in VR 2474 (ISU 79) (Figure 1).

The mRNA-junction sequences leading to mRNAs 3 and 4 of VR 2474 (ISU 79) and VR 2431 (ISU 1894) were determined to be GUAACC 89 nucleotides upstream of QLA 3 for sg 3 mRNA and UUCACC to 10 nucleotides upstream of QLA 4 for sg 4 mRNA (U.S. Serial No. 08 / 301,435, see also experiment 2 below). A comparison of the sequences of the isolates VR 2429 (ISU 22), VR 2430 (ISU 55) and VR 2431 (ISU 3927) with the isolates VR 2385, VR 2474 (ISU 79) and VR 2475 (ISU 1894) indicates that the sequences of mRNA binding to the leader for mRNAs sg 3 and 4 are conserved among the isolates from the US (Figure 1)

Figure 2 shows the alignment of the deduced amino acid sequences of the QLA 2 (A), QLA 3 (B), QLA 4 (C) and 47 QLA 5 (D) of the amino acid sequences encoded by QLAs 2 to 5. isolated from US VR 2429 (ISU22), VR 2430 (ISU 55) and VR 2431 (ISU 3927) with other known isolates of RSVRS. The sequence of VR 2385 is shown at the top and only the differences are indicated. Deletions are indicated by the (-) symbol. The proposed sequence of signal peptides in VLA (D) QLA 5 is underlined (Meulenberg et al., 1995). The three hypervariable regions with antigenic potentials in QLA 5 (D) were indicated by asterisks (*). The published sequences used in this alignment were VL (Meulenberg et al., 1993), VR 2385 (U.S. Patent Applications Serial Nos. 08 / 131,625 and 08 / 301,435), VR 2332, VR 2474 (ISU 79) and VR 2475 (ISU 1894) (U.S. Serial No. 08 / 301,435).

Based on its high basic amino acid content and hydrophilic nature, the translation product of QLA 7 is predicted to be the nucleocapsid protein (U.S. Patent Application Serial Nos. 08 / 131,625 and 08 And Meulenberg et al., 1993, Conzelmann et al., 1993, Mardassi et al., 1994). The QLA 6 product does not have a potential amino-terminal signal sequence and contains several hydrophobic regions which may represent the potential fragments of the transmembrane. Therefore, the product of QLA 6 was predicted to be protein M (U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435; Meulenberg et al., 1993; Conzelmann et al., 1993) . Computational analysis shows that the products encoded by QLAs 2 to 5 of U.S. isolates all have remaining hydropathic characteristics of membrane associated proteins. The translation products of QLAs 2 to 5 each contain a hydrophobic amino terminal 48. The N-terminal hydrophobic sequences may function as a signal sequence for each QLAs and may be involved in transporting the QLAs 2 to 5 to the endoplasmic reticulum of the infected cells. At least one additional hydrophobic domain was found in each of the CABs 2 to 5 at the carboxy terminus. These additional hydrophobic domains may function as membrane anchors.

The deduced amino acid sequences of QLAs 2 to 5 of the seven U.S. isolates examined varied considerably (Figure 2) indicating that most of the nucleotide differences observed in Figure 1 are not silent mutations. The identity of the amino acid sequences between VR 2385, VR 2332, VR 2429 (ISU 22), VR 2430 (ISU 55), VR 2474 (ISU 79) and VR 2475 (ISU 1894) was 95-99% in QLA 2, 90-9% in QLA 3, 94-98% in QLA 4 and 88-97% in QLA 5 (Table 2).

Again, the less virulent isolate VR 2431 (ISU 3927) showed more variation with other isolates from the US in QLAs 2 to 4 (Figure 2 and Table 2) than in QLAs 5 to 7 (U.S. 08 / 301,435 and Table 2). VLA QLA 2 to 5 share only 57-61%, 55-56%, 65-67%, and 51-55% amino acid sequence identity to the QLAs of the U.S. isolates, respectively (Table 2). Eliminations or insertions were found throughout the ALF 2 to 5 compared to the isolates of European and UV VL (Figure 2). Comparison of the sequences of the QLA 5 product showed that the N-terminal region of QLA 5 is extremely variable, both (a) between isolates from the US and VL as well as (b) among the various isolates from the US (Figure 2D) . In VL, the first 32-33 amino acid residues of QLA 5 may represent the signal sequence (Meulenberg et al., 1995; Figure 2D). Therefore, the potential signal sequence of QLA 5 in all isolates of the RSVRS is very heterogeneous. This heterogeneity is not due to any host immune system selection pressure because the signal peptide will be cleaved and will not be present in the mature virions.

Three more hypervariable regions were also identified by comparison to the amino acid sequences of QLA 5 of all available RSRS isolates (Figure 2D). The amino acid variations in these three regions are significant and are not structurally conserved (Figure 2D). Computational analysis indicates that all three hypervariable regions are hydrophilic and antigenic. Thus, it is likely that these regions will be exposed to the viral membrane and under selection pressure of the immune response of the host. However, further experiments may be required to confirm the specific functions of these hypervariable regions as antigenic determinants on the envelope protein of QLA 5.

Phylogenetic relationship among U.S. RSVRS isolates:

It has previously been shown that the U.S. RSRRS and the RSVR of Europe represent two distinct genotypes, based on analysis of the M and N genes (U.S. Serial No. 08 / 301,435). To determine the phylogenetic relationships of the U.S. RSRSRS isolates, from SEQs 2 to 7 of seven U.S. RSRSRS isolates shown in Figures 1 and 2, were first aligned with program 50

GeneWorks (Intelligenetics, Inc.) The PAUP program (David L. Swofford, Illinois Natural History Survey, Champaign, IL) was then used to construct the phylogenetic tree illustrating the relationship between U.S.

The phylogenetic tree of Figure 3 was constructed by processes with maximum parsimony with the help of the PAUP program package, version 3.1.1. We found the branch with the shortest length (the most parsimonious) implementing an exhaustive search option. The lengths of the branches (number of amino acid substitutions) are given above each branch. The sequences used in the analysis are VL, VR 2385, VR 2332, VR 2474 (ISU 79) and VR 2475 (ISU 1894). The phylogenetic tree indicates that there are at least three groups of variants (or minor genotypes) within the major genotype of the EU-6 RSVRS less virulent EU RSVRS, VR 2431 (ISU 3927), forming a distinct branch of the other isolates of the US (figure 3). Isolates VR 2429 (ISU 22), VR 2474 (ISU 79), VR 2475 (ISU 1894) and VR 2332 form another branch, which represents a second minor genotype. The third minor genotype is represented by isolates VR 2430 (ISU 55) and VR 2385 (Figure 3). A very similar tree was also obtained by analyzing the last 60 nucleotides of LFA 1b of the seven U.S. isolates shown in Figure 1 (data not shown). The topology of an identical tree was also generated by the process of unweighted pairs of arithmetic mean (PGNPPMA) using the GeneWorks program (data not shown).

In summary, different genotypes of RSVRS were confirmed and were better explained. Fifty-one at least three genotypes were identified within the main genotype of the United States RSRRS, based on an analysis of the sequence of QLAs 2 to 7. Genetic variations not only between the RSVR of Europe and the RSVR of the US, but also between the isolates from the US RSVRS were also also confirmed, indicating the heterogeneous nature of RSVRS. The less virulent U.S. RSVS isolate, VR 2431 (ISU 3927) unexpectedly has large sequence variations in QLAs 2 to 4, as compared to other U.S. isolates.

Table 2: Identities of nucleotide sequences and deduced amino acid sequences (%) of VSRRS ALS 2 to 5 VR2430 VR2434 VR2432 VR2438 VLA2475 VR2431 QLA 2 VR2385 (ISU22) (ISU55) (ISU79) (ISU3994) (ISU3994) VR2332 VL VR2385 * 97 96 96 95 91 98 58 VR2429 97 ★ 96 98 96 93 99 59 (ISU22) VR2430 98 97 96 95 91 97 61 (ISU55) VR2474 96 97 97 96 91 98 60 (ISU79) 52 (continued) QLA 2 (ISU3927) VR2332 VL VR2475 96 97 96 96 kk 93 96 57 (ISU1894) VR2431 94 94 94 93 93 kk 93 58 (ISU3927) VR2332 97 98 97 VR2432 (ISU3994) VR2432 (ISU3994) VR2432 98 97 94 59 VL 65 66 66 67 66 65 66 kk QLA 3 VR2385 kk 91 94 92 90 87 91 55 VR2429 92 kw 93 96 96 88 98 56 (15U22) VR2430 94 93 kk 94 93 87 94 56 (ISU55) VR2474 94 (ISU7927) VR2432 93 98 94 97 97 90 kw 56 VL 64 63 62 63 63 61 (ISU7927) VR2432 93 98 94 97 97 90 kk QLA4 VR2385 kk 94 96 94 95 83 94 66 VR2429 93 kw 94 97 99 93 98 66 (ISU22) VR2430 96 94 kk 96 96 93 95 67 (ISU55) VR2474 93 97 94 kk 98 92 96 66 (ISU79) VR2475 92 98 94 96 kk 93 98 66 (ISU1894) VR2431 91 93 92 91 91 92 67 (ISU3927) VR2332 94 99 95 97 98 92 kw 65 VL 66 66 63 65 66 65 65 kk QLA 5 VR2385 kw 90 91 88 89 91 89 54 VR2429 93 kw 90 94 96 92 97 52 (ISU22) VR2430 94 92 kw 89 89 90 89 51 (ISU55) VR2474 91 95 91 kk 95 89 94 53 (ISU79) VR2475 92 97 90 94 91 96 53 (ISU1894) VR2431 91 93 91 91 91 k 91 91 (ISU3927) VR2332 93 98 91 95 97 92 kk 53 53 VR2429 VR2430 VR2434 VR2475 VR2431

ORF 2 VR2385 (ISU22) (ISU55 (ISU79) (1SU1894) (ISU3927) VR2332 VL VL 63 63 63 61 62 63 63 t7 ~

Note: Comparisons of amino acid sequences are presented in the upper right half and comparisons of nucleotide sequences are presented in the lower left half EXPERIMENT 2

During the replication of the RSVRS, six subgenomic mRNAs (sg mRNAs) were synthesized in addition to the genomic RNA. These mRNAs were characterized in this experiment.

SRS rsRNA mRNAs form a nested set at the 3 'common end in the cells infected by RSRRS. Each of these sg mRNAs is polycistronic and contains multiple open reading frames, except for sg7 mRNA (as demonstrated by the Northern blot analysis where QLA-specific probes were used). The sg mRNAs were not packaged in the virions and only genomic RNA was detected in the purified virions, suggesting that the RSVR encapsidation signal is probably situated in the QLA 1 region. The number of sg mRNAs in the cells infected by the RSVRs varies among the isolates from VSRRS with different virulence. It has been shown in experiment 1 above that an additional species of sg mRNA in some of the RSVRS isolates derived from the sequence upstream of QLA 4 and had been designed as sg 4-1 mRNA.

The mRNA-junction sequences leading to the mRNAs sg 3 and 4 of the isolates VR 2474 (ISU79) and VR 2475 (ISU 1894) as well as the sg 4-1 mRNA of VR isolate 2474 (ISU 79) contained a common element of the six nucleotide sequence, T (G) TA (G / C) ACC. Analysis of the 54 genomic RNA sequences of these two U.S. isolates and their comparison with Lelystad (VL) virus revealed the heterogeneity of the mRNA junction sequences at the leader among the various isolates of the RSVRS. The number, locations, and sequences of the mRNA-to-leader junction regions varied between the isolates from the US and VL as well as from isolates from the US The last three nucleotides, ACC, from the mRNA-to-leader junction sequences are invariant . Variations were found in the first three nucleotides.

By comparing the 5 'end sequence of sg 4-1 mRNA with the genome sequence of VR 2474 (ISU 79) and VR 2475 (ISU 1894), that a single nucleotide substitution, of T in the VR 2475 (ISU 1894) to C in VR 2474 (ISU 79), led to a novel mRNA-binding sequence at the leader in ISU 79, and therefore to an additional species of sg mRNA (sg 4-1 mRNA). A small QLA, designated QLA 4-1, with a coding capacity of 45 amino acids, was identified at the 5 'end of mS4-1 mRNA.

MATERIALS AND PROCESSES

Viruses and cells. VR 2429 (ISU 22), VR 2430 (ISU 55), VR 2474 (ISU 79), VR 2475 (ISU 1894) and VR 2431 (ISU 3927) were isolated from pig lungs obtained from different farms in Iowa. A continuous cell line, ATCC CRL 11171, was used for isolation and development (culturing) of the viruses. These isolates were cloned biologically for 3 plaque purification passages and growth in CRL cells 11171. All of the virus isolates used in this study were in the seventh passage. 55 VR 2429 (ISU 22) and VR 2474 (ISU 79) are highly pathogenic and produce between 50 and 80% consolidation of lung tissues in piglets at 5 weeks In contrast, the VR 2430 (ISU 55), VR 2475 (ISU 1894) and VR 2431 (ISU 3927) are low in age, cesarean-born, devoid of colostrum, infected in the laboratory and autopsied on the 10th day after inoculation. pathogenicity and produced only between 10 and 25% of the and lung tissues in the same experiment (U.S. Patent Application Serial Nos. 08 / 131,625 and 08 / 301,435).

Preparation of total intracellular RNAs specific for the virus, poly (A) * RNA and virion RNA. Confluent monolayers of CRL 11171 cells with different RSRSV isolates were infected in the seventh passage with a multiplicity of infection (m.d.i.) of 0.1. The total intracellular RNAs specific for the RSGRS were isolated from the cells infected with the RSGRS by a standard procedure with guadinine isothiocyanate (Stratagene). Poly (A) * RNA was enriched from total intracellular RNAs by cellulose-oligo dT column affinity chromatography (Invitrogen).

For the isolation of the RNA from the virus of the RSVRS virus, confluent CRL 11171 cells were infected with the RSVRS isolate VR 2431 (ISU 3927) with an m.d.i. of 0.1. When more than 70% of the infected cells had a cytopathic effect, the cultures were frozen and thawed three times and the culture medium purified by centrifugation at 1200 x g for 20 minutes at 4 ° C. The virus was then precipitated with polyethylene glycol and subsequently purified by centrifugation with a cesium chloride gradient as described in U.S. Serial No. 56 08 / 131,625. The purified virus was treated with Rnase A at a final concentration of 20æg / ml for 90 minutes at 37øC. The virus was then agglomerated and the RNA isolated from the virion using the standard procedure with guanidinium isothiocyanate (Stratagene). Synthesis of the cDNA and polymerase chain reaction.

The cDNA was synthesized from the total intercellular RNAs by reverse transcription using random primers and amplified by polymerase chain reaction (PCR-TI) as described previously (Meng et al., 1993, J. Vet. , 5: 254-258).

&Quot; Northern blot analysis ". Ten μg of total intercellular RNAs from virus infected cells and from falsely infected cells were used for each column on a formaldehyde-containing agarose gel. For the separation of the poly (A) * RNA and the virion RNA, fifteen æg of the virion RNA and 0.2 æg of the poly (A) * RNA were loaded per column. The RNA was denatured with formaldehyde according to a standard procedure (Sambrook et al., &Quot; Molecular Cloning: A Laboratory Manual ", 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The RNA was separated by electrophoresis, transfer of gel RNA to a matrix (blotting) and hybridization, as described in U.S. Serial No. 08 / 131,625. In some experiments, glyoxal-DMSO agarose gels have also been developed as described in U.S. Patent Application Serial Nos. 08 / 131,625.

For the preparation of the probes, a specific cDNA fragment was generated for each of the QLAs 1 to 7 by CT-PCR with specific primers for the QLA. The 57 primers were designed in such a way that each primer pair amplified only a specific fragment of a given QLA and the overlap, in the vicinity of the QLAs, is not included in any of these cDNA probes. Primer pairs for generating the cDNA probes representing the 1 to 7 QLAs are IM729 / 1M732 for QLA1, IM312 / 1M313 for QLA2, XM1022 / 1M258 for QLA3, XM1024 / XM1 023 for QLA4, PP287 / PP286 for QLA 5, PP289 / XM780 for QLA 6 and PP285 / PP284 for QLA 7 (Table 3).

Cloning, sequencing and analysis of nucleotide sequences. The primers for PCR-TI were designed based on the sequences of VR 2385 isolated from the RSVRS, which amplified the regions that encode the entire protein from QRSs 2 to 5 of the RSVRS isolates VR 2474 (ISU 79) and VR 2475 (ISU 1894). Primers JM259 and JM260 were used for the amplification of QLAs 4 and 5 and XM992 and XM993 for the amplification of QLAs 2 and 3 (Table 3). Single restriction sites (EcoRI and BamHI) were introduced at the ends of the PCR products, thus allowing a cassette approach for the replacement of these QLAs.

The PCR products of QLAs 2-3 and QLAs 4-5 of VR 2474 (ISU 79) and VR 2475 (ISU 1894) were digested with EcoRI and BamHI, then purified and cloned into pSK + vectors as described previously (Meng et al., 1993, 1. Vet. Diagn. Invest., 5: 254-258). The plasmids containing the viral inserts were sequenced with a conventional automated DNA sequencer (Applied Biosystem, Inc.). At least three cDNA clones, which represented the entire sequence of QLAs 2 to 5 of each of the virus isolates, were sequenced with universal and reverse primers, as well as other virus-specific sequencing primers (XM969, XM970, XM1006 , XM078 and XM077 (see Table 3).

To determine the mRNA-junction sequences to the leader of sg 3 and 4 mRNAs and sg 4-1 mRNAs, primer pairs IM755 and DP586 (Table 3) were used for PCR-TI to amplify the corresponding terminal sequences 5 '. The resulting PCR products were purified and sequenced by direct PCR sequencing using the XMD77 and XM141 virus specific primers (Table 3). Sequences were combined and analyzed with MacVector (International Biotechnologies, Inc.) and GeneWorks (IntelliGenetics, Inc.) computer software programs.

Oligonucleotides. The synthetic oligonucleotides used in this study are summarized in Table 3. These oligonucleotides were synthesized as single-stranded DNA using an automated DNA synthesizer (Applied Biosystem) and purified by high performance liquid chromatography (HPLC).

RESULTS

The sg mRNAs are not packaged in the PRRSV virions.

To determine if the ssRNA mRNAs of the RSVRS are packaged, the virions of the RSVRS isolate VR 2431 (ISU 3927) were purified by centrifugation with a CsCl gradient. Purified virions were treated with RNase A prior to pelleting the virions and extracting the RNA to remove any RNA species that may have adhered to the surface of the virions. RNAs from the RNase A-treated virions were separated together with the total intracellular RNAs of the VR 2431 isolate (ISU 3927) from the cells infected with 59 RSVRS on a formaldehyde gel and hybridized with a probe generated from the terminal sequence 3 'of the virus genome by PCR with primers PP284 and PP285 (U.S. Serial No. 08 / 131,625; Table 3).

Genomic RNA was detected only in the purified virions of the RSVRS isolate VR 2431 (ISU 3927) (Figure 4) and undetectable amounts of sg mRNAs were observed in the purified virions even three weeks post-exposure. In contrast, seven species of sg mRNAs, in addition to genomic RNA, were detected in cells infected with VR 2431 (ISU 3927) (figure 4). Similar results were observed with two other U.S. RSRVS isolates, VR 2430 (ISU 55) and VR 2474 (ISU 79).

Variation in the number of sg mRNAs between the isolates of the U.S. RSVRS with different virulence. All known arterviviruses prior to the present invention, including EU RSVRS and RSVRS of Europe, were shown to produce six sg mRNAs, except for three variants of VDHL (VDHL-P, VDHL-a and VDHL-v), which synthesize seven mRNAs sg. However, a set of six sg mRNAs is produced in the VDHL-C strain.

To compare if there are many variations in sg mRNAs between U.S. RSVS isolates, confluent monolayers of CRL 11171 cells were infected with five isolates other than the U.S. RSVRS with different virulence at one m.d.i. of 0.1. Total intercellular RNAs were isolated from the cells infected by the virus 24 h after infection. A cDNA fragment was generated from the 3 'end of the viral genome by PCR with primers PP284 and PP285 (Table 3). The cDNA fragment 60 was labeled with32P-dCTP (32P-radiolabeled deoxycytosine triphosphate) by the extension process of the random primers and hybridized to the total intercellular RNAs (separated on a formaldehyde gel).

Analyzes of the RNAs showed that a nested set of the six or more sg mRNAs, in addition to genomic RNA, was present in cells infected with one of the five U.S. RSVRS isolates with different virulence (Figure 5). Similar results were obtained when total intracellular RNAs were separated by agarose gel electrophoresis with glyoxal / DMSO (dimethylsulfoxide). Isolates from VRSR, VR 2430 (ISU 55), VR 2474 (ISU 79) and VR 2431 (ISU 3927) produced seven easily distinguishable sg mRNAs, whereas VR 2429 (ISU 22) and VR 2475 (ISU 1894) isolates produced six MRNAs sg (figure 5). The U.S. RSVRS isolate, VR 2385, also produced six sg mRNAs (U.S. Application Serial Nos. 08 / 131,625). An additional species of sg mRNA was located between mRNAs sg 3 and 4 and was designated sg 4-1 mRNA. The sg mRNAs, if any, differ little in size among the five RSRVS isolates (figure 5). It appears, however, that there is no correlation between pneumovirulence and the number of sg mRNAs observed in these five isolates. Sg 4-1 mRNA is not vim interfering defective RNA and is not the result of nonspecific binding of the probes to ribosomal RNAs. A wide variety of interfering defective RNA (D1 RNA) of different sizes was generated in coronaviruses when VHR was successively passed through the tissue culture with an m.d.i. high. D1 RNAs were also observed in cells infected with torovirus during passage without dilution. For this reason, we investigated the possibility that the SRS 4-1 sRNA mRNA is a D1 RNA.

To exclude this possibility, the original virus stock of the RSVRS isolate, VR 2474 (ISU 79), which produced the additional species of sg 4-1 mRNA, was passaged four times in CRL 11171 cells with different m.d.i. 0.1, 0.01 and 0.001, respectively. In a control experiment, four passages were made, without dilution, of the original virus stock of VR 2474 (ISU 79). After the four passages, total intracellular RNAs were isolated from the cells infected with the virus and analyzed by " Northern blot ", with the same probe generated from the 3 'end of the viral genome.

Analyzes of sg mRNAs showed that additional species of sg 4-1 mRNA were still present in all RNA preparations with different m.d.i as well as in the RNA preparations of the undiluted passages (Figure 6A). In addition, there was no interference or reduction in the synthesis of other sg mRNAs in the presence of sg 4-1 mRNA, as is usually the case with Dl RNA.

VHR D1 RNAs were shown to disappear after two passages at a high dilution. Therefore, if the original virus stock of VR 2474 (ISU 79) contained D1 RNA, then D1 RNA should disappear after four passages with high dilution. Previous experimental data suggest that, unlike D1 RNA, replication of sg 4-1 mRNA is independent of the amount of the standard virus. It follows that sg 4-1 mRNA is not a D1 RNA. 62

In the analysis by " Northern blot " of the total intracellular RNAs, the probes may bind in a non-specific manner to the 18S and 28S ribosomal RNAs, which are abundant in the cytoplasmic RNA preparations. Alternatively, abundance of the ribosomal RNAs may cause the retardation of specific sg mRNAs of the virus that may migrate corrugated with the ribosomal RNAs in the gel.

Two additional bands were observed due to non-specific binding of probes to ribosomal RNAs in VL-infected cells and in VDHL-infected cells. Therefore, it is possible that ssRs 4-1 mRNAs are due to nonspecific binding of the probes to ribosomal RNAs.

To exclude this possibility, the polyadenylated RNA was isolated from the total intracellular RNAs of CRL 11171 cells infected with any of the RSVRS isolates, VR 2430 (ISU 55) and VR 2474 (ISU 79). Both VR 2430 (ISU 55) and VR 2474 (ISU 79) produce both additional species of sg 4-1 mRNA (Figure 5). Northern blot analysis " of polyadenylated RNA showed that additional species of sg 4-1 mRNA in cells infected by either of these two isolates were still present (Figure 6B), indicating that sg 4-1 mRNA is not due to nonspecific binding of the probes to the RNAs ribosomal.

The sg mRNAs represent a nested set at the 3 'common end and the sg 4-1 mRNA is derived from a sequence upstream of the LFA4. Six sg mRNAs, in addition to the genomic RNA, were detected in the cells infected by VR 2385 using a probe of cDNA from the 3 'end of the viral genome (U.S. Application Serial No. 08 / 131,625). Thus, such as Berne viruses (VBE), VDHL, VAE, coronavirus, and VL, replication of the RSVRS of the US also requires the synthesis of a nested set at the 3 'common end of sg mRNAs (patent applications U.S. Serial Nos. 08 / 131,625 and 08 / 131,435).

To analyze these sg mRNAs in more detail, seven cDNA fragments specific for each of the 1 to 7 CFA's were amplified by PCR. Primer designs for PCR were based on the sequence of VR 2385. The sequences and locations of the primers, IM729 and IM782 for QLA1, IM312 and IM313 for QLA2, XM1022 and IM258 for QLA3, XM1024 and XM1023 for QLA 1, PP286 and PP287 for QLA 5, PP289 and XM780 for QLA 6 and PP284 and PP285 for QLA 7 and the 3 'non-coding region (RNC) are shown in Table 3. The primers were designed such that each set of primers will amplify only one fragment of a particular QLA and the overlapping sequences between the QLAs of the neighborhood are not included in any fragment. Thus, each of these seven DNA fragments represents only one particular LFA, except for fragment 7, which represents both LFA 7 and 3'-RNC. oo

These seven DNA fragments were labeled with P-dCTP and Northern blots were hybridized from the total intracellular RNAs extracted from the infected cells with either of the two isolates from the RSVRS of the EU, VR 2475 (ISU 1894) and VR 2474 (ISU 79). Included as a control were total intracellular RNAs isolated from a falsely infected CRL 11171 cell line.

Northern blot analyzes " showed that probe 1, generated from QLA 1b, hybridized only to genomic RNA. Probes 2 to 7 each hybridized with a further RNA species in addition to the genomic RNA (Figure 7). The results indicate that a nested set, at the 3 'common end, of six or more sR mRs from VR 2475 (ISU 1894) or from VR 2474 (ISU 79) on the RSVRS infected cells (Figures 7A and 7B) , with the smallest RNA of the 3 'terminal (sg7 mRNA) encoding the 7DNA. All ssRNAs of the EURSSR contained the 3' end of the genomic RNA, but extended at various distances towards the 5 'end of the genome, depending on the size of sg mRNA. The mRNA sg 4-1 of the RSVRS isolate VR 2474 (ISU 79) hybrid with probes 4 to 7, but not with probes 1, 2 and 3 (Figure 7B), suggesting that sg 4-1 mRNA contains the QLAs 4 to 7 well with RNC 3 '. Accordingly, sg 4-1 mRNA is generated from the upstream sequence of QLA 4. Replacement of a single nucleotide leads to the acquisition of additional species of sg 4-1 mRNA. The hybridization data of the " Northern blot " showed that sg 4-1 mRNA is derived from a sequence upstream of QLA 4 (Figure 7B). To determine the exact location and mating sequence of the mRNA to the sg 4-1 mRNA leader, a set of primers, IM755 and DP586 (Table 3) was designed. The forward primer IM755 was prepared based on the 3 'end of the VR 2385 leader sequence and the DP586 reverse primer is located on the QLA 4 (Table 3).

PCR-TI was performed with primers IM755 and DP586 using the total intracellular RNAs of cells infected by either VR 2475 (ISU 1894) or VR 2474 (ISU 79) isolates. VR 2474 (ISU 79) produces ss 4-1 mRNA, but VR 2475 (ISU 1894) does not (figure 5). CT-PCR was prolonged for a further 30 seconds to preferably amplify the short fragments representing the 5 'terminal sequences of the mRNAs sg 3, 4 and 4-1. Analysis of the PCR-TI products showed that two fragments, about 1.1 kb and 0.45 kb in size, had been amplified from the total RNAs of the cells infected with the virus VR 2475 (ISU 1894) (Figure 8A). These two fragments represent portions of the 5 'end of the mRNAs sg 3 and 4, respectively. In addition to the two fragments observed in the VR 2475 isolate (ISU 1894), a third fragment of about 0.6 kb representing the 5 'end of the sg 4-1 mRNA was also amplified from the total cell RNAs infected with VR 2474 (ISU 79) (Figure 8A).

To determine the mRNA-junction sequences to the mRNA leader sg 3, 4 and 4-1, the PCR products of VR 2474 (ISU 79) and VR 2475 (ISU 1894) were purified with an agarose gel using a GENECLEAN kit (Bio 101, Inc.) and sequenced directly with an automated DNA sequencer (Applied Biosystems). The primers used for 5 'end sequencing of the PCR products (XM141 and XM077, Table 3) were designed based on the genomic sequences of VR 2474 (ISU 79) and VR 2475 (ISU 1894) (Figure 9 ). The mRNA binding sequences were determined on the leader (where the leader joins the mRNA body during mRNA synthesis) of the mRNAs sg 3, 4 and 4-1 of the two isolates of the RSVRS of the EU, by comparing the sequences from the 5 'end of the sg mRNAs and the genomic RNA of the two isolates (Figure 8B).

Junction mRNA sequences to the leader of the mRNAs sg 3 and 4 of VR 2475 (ISU 1894) and VR 2474 (ISU 79) were identical. For sg 3 mRNA, the leader-binding sequence 66 (GUAACC) is located 89 nucleotides upstream of QLA 3. For mRNA sg 4, UUCACC is located 10 nucleotides upstream of QLA 4 (Figure 8B and Figure 9). The mRNA-junction sequence leading to mRNA sg 4-1 of VR 2474 (ISU79) is UUGACC, located 236 nucleotides upstream of QLA 4 (Figures 8B and 9). Sequence alignment of VR 2474 (ISU 79) and VR 2475 genomic sequences (ISU 1894) shows that the substitution of a single nucleotide, from T in VR 2475 (ISU 1894) to C in VR 2474 (ISU 79), leads to the acquisition of an additional mRNA-binding sequence to the leader, UUGACC, in VR 2474 (ISU 79) (Figures 8B and 9). Thus, an additional species of sg (4-1) mRNA is formed (Figure 5). In addition to QLAs 4 to 7 contained in mRNA 4, sg 4-1 mRNA contains at the 5 'end an additional small QLA (QLA 4-1) with a coding capacity of 45 amino acids (Figure 9). This small QLA stops only one nucleotide before the QLA start codon 4. Analysis of the QLA sequences 2 to 7 of the two U.S. isolates reveals heterogeneity of the mRNA junction sequences to the leader. Clones 2 to 5 of VR 2474 (ISU 79) and VR 2475 (ISU 1894) were cloned and sequenced (see experiment 1 above). VR 2474 (ISU 79) yields seven easily distinguishable sg mRNAs, whereas VR 2475 (ISU 1894) yields six distinguishable sg mRNAs (Figures 5 and 7). At least three clones of the cDNA in a given region of the BLAs 2 to 5 were sequenced for each virus isolate, using both universal and reverse primers, as well as primers specific for XM969, XM970, XM1006, XM07 8 and XM077 virus (Table 3 ). The sequences of QLAs 6 and 7 of VR 2475 (ISU 1894) and VR 2474 (ISU 79) are described in U.S. Serial No. 08 / 301,435. Sequence analysis showed that QLAs 2 to 7 of VR 2474 (ISU 79) and VR 2475 (ISU 1894) overlap each other, except for a non-coding region of 10 nucleotides between QLA 4 and QLA 5 This same observation was made earlier for VR 2385 (U.S. Application Serial No. 08 / 301,435). This is very unusual, since all members of the proposed Arterivirus family, including VL, contain overlapping QLAs. However, the QLAs of the coronaviruses are separated by non-coding intergenic sequences. Thus, the U.S. RSVR appears to be somewhat similar to coronaviruses in terms of the injunction regions of the genomic organization of QLAs 4 and 5. QLA 2 of VR 2475 (ISU 1894) was a longer amino acid than that of ISU 7 9 (Figure 9). The termination codon of QLA 2, TAG, was changed to TGG in VR 2475 (ISU 1894) immediately followed by a new termination codon (TGA) in VR 2475 (ISU 1894) (Figure 9). The sizes of other QLAs of VR 2474 (ISU 79) and VR 2475 (ISU 1894) were identical (Figure 9). There were no deletions or insertions in QLAs 2 to 7 of these isolates. However, numerous substitutions are present throughout the entire sequence of QLAs 2 to 7 between VR 2474 (ISU 79) and VR 2475 (ISU 1894) (Figure 9). The number and locations of the predicted or predicted mRNA-to-leader junction sequences range from VR 2475 (ISU 1894) to VR 2474 (ISU 79) (Figure 9). In addition to the normal mRNA 4-to-leader sequence, TTCACC, 10 nucleotides upstream of QLA 4, there was also an additional 4-1 mRNA binding sequence to the leader, (TTGACC) located 236 nucleotides upstream of QLA 4 in VR 2474 (ISU 79) (Figure 9). The junction sequences of

MRNAs sg 4 and 4-1 to the leader were separated by 226 nucleotides, which correlates with the estimated sg 4 and 4-1 mRNA sizes observed in the " Northern blot " (Figure 5) and CT-PCR amplification (Figure 8A). The mRNA 3-to-leader junction sequence is identical between VR 2475 (ISU 1894) and VR 2474 (ISU 79) GTAACC, located 89 nucleotides upstream of QLA 3. The predicted mRNA junction sequences at the leader are mRNAs sg 2 and 6 of VR 2475 (ISU 1894) and VR 2474 (ISU 79) were also identical (Figure 9).

However, the predicted junction sequences of mRNA 5 at the leader of VR 2475 (ISU 1894) and VR 2474 (ISU 79) are different (Figure 9). There are 3 potential mRNA 5 junction sequences to the leader for VR 2475 (ISU 1894) and VR 2474 (ISU 79) (GCAACC, GAGACC and TCGACC, located 55, 70 and 105 nucleotides upstream of QLA 5, respectively). Two potential mRNA 5 binding sequences were also found to be leader to VR 2475 (ISU 1894) (GAAACC and TCGACC, located 70 and 105 nucleotides upstream of QLA 5, respectively) (Figure 9). The differences were due to the two nucleotide substitutions in the predicted mRNA 5 junction sequences at the leader of these isolates (Figure 9).

In addition to the 7 mRNA binding sequence to the leader, 15 nucleotides upstream of QLA 7, an additional leader mRNA 7 junction sequence (ATAACC) was located, located 129 nucleotides upstream of QLA 7 in each of these two isolated (Figure 9). However, mRNA sg, which corresponds to this additional mRNA-7 junction sequence to the leader, is not clearly distinguished from the abundant mg7 mRNA which produced a diffuse broad band in the " Northern blot " (Figures 5, 6 and 7).

Variations in the number and locations of the mRNA-to-leader junction sequences between the VL and the two US isolates analyzed in this experiment were also found by comparing the mRNA-mRNA-binding sequences to the leader of the VL with those of the two isolates from the EU VR 2475 (ISU 1894) and VR 2474 (ISU 79). Considering these data together, they indicate that SRS rsRNA mRNAs are polymorphic, that the exact number and size of mRNAs sg depends on the specific PRRSV isolate analyzed. However, a nested set of the six sg mRNAs most likely reflect the standard organization and transcription of the arterivirus genome.

Table 3. Synthetic oligonucleotides used in the experiment 2 70

Name of oligo. Sequence Location (nucleotides) " Polarity b IM729 5'-GACTGATGGTCTGGAAAG-3 'QLAlb, -507 to -490 upstream of QLA2 + IM782 5'-CTGTATCCGATTCAAACC-3' QLAlb, -180 to-163 upstream of QLA2-1M312 5'-AGGIIGGCIGGIGGICII-3 'QLA2 , 131 to 146 downstream of QLA2 + IM313 5'-TCGCTCACTACCTGTTTC-3 'QLA2, 381 and 398 downstream of QLA2-XM1022 5'-TGTGCCCGCCTTGCCTCA-3' QLA3, 168 to 175 downstream of QLA3 + IM2 68 5'- AAACCAATTGCCCCCGTC-3 'QLA3, 520 to 537 downstream of QLA3 XM1024 5'-TATAT CAC TG T CACAGCC-3' QLA4, 232 to 249 downstream of QLA4 + XM1023 5'-CAAATTGCC70ACAGAATG-3 'QLA4, 519 to 536 downstream of QLA4-PP287 5'-CAACTTGACGCTATGTGAGC-3 'QLA5,129a 148 downstream of QLA5 + PP286 5'-GCCGCGGAACCATCAAGCAC-3' QLA5, 538 to 667 downstream of QLA5-PP289 5'-GAC T GC TAGGGC TTCT GCAC-3 'QLA6,1191 and 138 downstream of QLA6 + XM780 5'-CGTTGACCGTAGTGGAGC-3' QLA6, 416 to 433 downstream of QLA6-PP283 5'-CCCCATTTCCCTCTAGCGACTG-3 'QLA7, 157 to 178 downstream of QLA7 + PP284 '-CGGCCGTGTGGTTCTCGCCAAT-3' 3'RCN, -27 to -6 a (A) -JM2 60 5'-GGGGAATTCGGGATAGGGAATGTG-3 'QLA3, 338 to 356 downstream of QLA3 + JM25 9 5'-GGGGATCCTTTGTGGAGCCGT-3' QLA6, 34 to 52 downstream QLA6-XM9 93 5'-GGTGAATTCGTTTTTTTCCCTCCGGGC -3 'QLA1b, -53 to -35 upstream of QLA2 + XM9 92 5'-GGGGGATCCTGTTGGTAATAGI AGTCTG-3' QLA3, -50 to -34 upstream of QLA4-XM97 0 5'-GGTTTCACCTAGAATGGC-3 'QLA2, 522 to 530 downstream of QLA2 + XM96 9 5'-GATAGAGTCTGCCCTTAG-3 'QLA3, 443 to 460 downstream of QLA6-XM1006 5'-GCTTCTGAGATGAGTGA-3' QLA4, 316 to 332 downstream of QLA4 + XM0785 ' -CT GAGCAAT TACAGAAG-3 'OR F2, 202 to 210 downstream of QLA2 + XM0 7 7 5'-C7VACCACGCGT7V7VACACT-3' QLA3, 316 to 333 downstream of QLA3-IM755 5'-GACTGCTTTACGGTCTCTC-3 'Lider, 3' 'of the leader sequence + XM1141 5'-GATGCCTGACACATTGCC-3' QLA4, 355 to 372 downstream of QLA4-DP586 5'-CT GCAAGAC T CGAAC T GAA-3 'QLA4, 78 to 97 downstream of QLA4-a. Oligonucleotides were designed based on sequence data presented in this patent application and in U.S. patent applications Serial Nos. 08 / 131,625 and 08 / 301,435 b. Oligonucleotides complementary to genomic RNA with negative (-) polarities.

Lisbon, July 24, 2009 71

Claims (12)

A purified composition comprising a polynucleic acid encoding at least one polypeptide selected from: a protein having the sequence of ISU55 shown in Figure 2A, encoded by QLA 2 of the RSVAR isolate deposited with ATCC No. Access VR 2430; a protein having the sequence of ISU55 shown in Figure 2B, encoded by QLA 3 of VR isolate 2430; a protein having the sequence of ISU55 shown in Figure 2C, encoded by QLA 4 of VR 2430; a protein having the sequence of ISU55 shown in Figure 2D, encoded by QLA 5 of VR 2430, wherein the purified preparation is not the natural VR 2430 virus. A purified composition according to claim 1, wherein said polynucleic acid encodes a protein having the sequence of ISU55 shown in Figure 2D, encoded by VLA 2430 QLA 5. A purified composition which comprises a nucleic acid having the formula (V): wherein: â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒ (3): the genes encoding at least one polypeptide and optionally one or more genes operably linked to one another and which are selected from the group consisting of (a) a conventional label or a reporter gene, (b) genes encoding at least one polypeptide or a its antigenic fragment, encoded by a QLA 5 of an Iowa strain of the RSVRS; and (c) genes encoding at least one polypeptide or an antigenic fragment thereof encoded by a QLA 6 or a QLA7 of an Iowa strain of the RSVRS; ε, which is optionally present, represents a sequence of the 5 'terminal polynucleotide which provides a means for the operative expression of the κ polynucleotide; ζ represents a polynucleotide of formula KTV ACC, wherein K is T, G or U and V is A, G or C; T represents a polynucleotide at most about 130 nucleotides in length; and ξ, which is possibly present and operably linked to ε, represents a 3 'terminal polynucleotide sequence that does not suppress the operative expression of the κ polynucleotide, wherein the polynucleic acid is not comprised of the sequence GCAGGCTTT GCTGTCCTCC AAGACATGAG TTGCCTTAGG CATCGCAACT CGGCCTCTGA 60 GGCGATTCGC AAAGTCCCTC AGTGCCGCAC GGCGATAGGG ACACCCGTGT ATATCACTGT 120 CACAGCCAAT GTTACCGATG AGAATTATTT GCATTCCTCT GATCTTCTCA TGCTTTCTTC 180 TTGCCTTTTC TATGCTTCTG AGATGAGTGA AAAGGGATTT AAGGTGGTAT TTGGCAATGT 240 GTCAGGCATC GTGGCAGTGT GCGTCAACTT CACCAGTTAC GTCCAACATG TCAAGGAATT 300 TACCCAACGT TCCTTGGTAG TTGACCATGT GCGGCTGCTC CATTTCATGA CGCCCGAGAC 360 CATGAGGTGG GCAACTGTTT TAGCCTGTCT TTTTGGCATT CTGTTGGCAA TTTGAATGTT 420 TAAGTATGTT GGGGAAATGC TTGACCGCGG GCTGTTGCTC GCAATTGCTT 7TTTTGTGGT 480 GTATCGTGCC GTCTTGTTTT GTTGCGCTCG TCAGCGCCAA CGGGAACAGC GGCTCAAATT 540 TACAGCTGAT TTACAACTTG ACGCTATGTG AGCTGAATGG CACAGATTGG CTAGCTAATA 600 AATTTG ACTG GGCAGTGGAG TGTTTTGTCA TTTTTCCTGT GTTGACTCAC ATTGTCTCTT 660 ATGGTGCCCT CACTACTAGC CATTTCCTTG ACACAGTCGG TCTGGTCACT GTGTCTACCG 720 CTGGGTTTGT TCACGGGCGG TATGTTCTGA GTAGCATGTA CGCGGTCTGT GCCCTGGCTG 780 CGTTGATTTG CITCGTCATT AGGCTTGCGA AGAATTGCAT GTCCTGGCGC TACGCATGTA 840 CCAGATATAC CAACTTTCTT CTGGACACTA AGGGAAGACT CTATCGTTGG CGGTCGCCTG 900 TCATCATAGA GAAAAGGGGC AAAGTTGAGG TCGAAGGTCA CCTGATCGAC CTCAAAAGAG 960 TTGTGCTTGA TGGTTCCGCG GCTACCCCTG TAACCAGAGT TTCAGCGGAA CAATGGAGTC 1020 GTCCTTAGAT GACTTCTGTC AT GATAGCAC GGCTCCACAA AAGGTGCTCT TGGCGTTTTC 1080 TATTACCTAC ACGCCAGTGA TGATATATGC CCTAAAGGTG AGTCGCGGCC GACTGGTAGG 1140 GCTTCTGCAC CTTTTGGTCT TCCTGAATTG TGCTTTCACC TTCGGGTACA TGACATTCGT 1200 GCACTTTCAG AGTACAAATA AGGTCGCGCT CACTATGGGA GCAGTAGTTG CACTCCTTTG 1260 GGGGGTGTAC TCAGCCATAG AAACCTGGAA ATTCATCACC TCCAGATGCC GTTTGTGCTT 1320 GCTAGGCCGC AAGTACATTC TGGCCCCTGC CCACCACGTT GAAAGTGCCG CAGGCTTTCA 1380 TCCGATTGCG GCAAATGATA ACCACGCATT TGTCGTCCGG CGTCCCGGCT CCACTACGGT 1440 CAA CGGCACA TTGGTGCCCG GGTTAAAAAG CCTCGTGTTG GGTGGCAGAA AAGCTGTTAA 1500 ACAGGGAGTG GTAAACCTTG TTAAATATGC CAAATAACAC CGGCAAGCAG CAGAAGAGAA 1560 AGAAGGGGGA TGGCCAGCCA GTCAATCAGC TGTGCCAGAT GCTGGGTAAG ATCATCGCTC 1620 ACCAAAACCA GTCCAGAGGC AAGGGACCGG GAAAGAAAAA TAAGAAGAAA AACCCGGAGA 1680 AGCCCCATTT CCCTCTAGCG ACTGAAGATG ATGTCAGACA TCACTTTACC CCTAGTGAGC 1740 GTCAATTGTG TCTGTCGTCA ATCCAGACCG CCTTTAATCA AGGCGCTGGG ACTTGCACCC 1800 TGTCAGATTC AGGGAGGATA AGTTACACTG TGGAGTTTAG TTTGCCTACG CATCATAATG 1860 TGCAGCTGAT CCGCGTCACA GCATCACCCT CAGCATGATG GGCTGGCATT CTTGAGGCAT 1920 CCCAGTGTTT GAATTGGAAG AATGCGTGGT GAATGGCACT GATTGACATT GTGCCTCTAA 1980 GTÇACCTATT CAATTAGGGC GACCGTGTGG GGGTAAGATT TAATTGGCGA GAACCACACG GCGGAAATTA 2040 AAAAAAAAAA AA-purified preparation of 2062. according to claim 1, wherein said polynucleic acid encoding at least one reqião 3 hypervariable of a protein encoded by a QLA 5 of an Iowa strain of RSVR. A polynucleic acid characterized in that it encodes at least one polypeptide comprising at least one hypervariable region of QLA 5 of an Iowa strain of RSVRS selected from: Hypervariable Region 1: SNDSSSH, SSSNSSH, SANSSSH, HSNSSSH, SNSSSSH, NNSSSSH , NGGDSST (Y); Hypervariable Region 2: ANKFDWAVET, ANKFDWAVEP, AGEFDWAVET, ADKFDWAVEP, ADRFDWAVE P, SSHFGWAVET; Hypervariable region 3: LTCFVIRFA, LICFVIRFT, LACFVIRFA, LTCFVIRFV, LTCFIIRFA, FICFVIRFA, FVCF-VIRAA. A polynucleic acid according to claim 5, wherein the polypeptide further comprises amino acid sequences of a QRSV of the RSVRS. A purified polypeptide, characterized in that it is encoded by polynucleic acid according to any one of claims 1 to 6. Vaccine, characterized in that it comprises (a) an effective amount of the polypeptide according to claim 7 for protecting pigs against the reproductive and respiratory syndrome of swine and (b) a physiologically acceptable carrier. Vaccine, characterized in that it comprises (a) an effective amount of polynucleic acid according to any one of claims 1 to 7 to protect pigs against the reproductive and respiratory syndrome of the pigs and (b) an acceptable carrier under the physiological point of view. Antibody, characterized in that it specifically binds to the polypeptide according to claim 7. An antibody according to claim 10, wherein said antibody is a monoclonal antibody Use of an antibody according to claim 10 or 11 for the preparation of a medicament for treating pigs suffering from reproductive and respiratory syndrome of swine. A diagnostic kit for assaying a reproductive and respiratory syndrome virus of the swine, comprising the antibody according to claim 10 or claim 11 and a diagnostic agent indicating the positive immunological reaction with said antibody. A process for the production of a polypeptide, characterized in that it expresses the polynucleic acid according to any one of claims 1 to 6 in an operational expression system. An expression vector, characterized in that it contains the polynucleic acid according to any one of claims 1 to 6, under the operational control of a promoter. Lisbon, July 24, 2009 5